KR20120091251A - Sorf constructs and multiple gene expression - Google Patents

Abstract

Aspects of the invention relate to vector constructs and methods for the expression of polypeptides comprising multimeric products, such as therapeutic antibodies. Special constructs allow the production of expression products from a single transcriptional translation frame (sORF). One embodiment provides an isolated or purified expression vector for producing one or more recombinant protein products comprising a single transcription translation frame insert; The insert comprises a signal peptide nucleic acid sequence encoding a signal peptide; A first nucleic acid sequence encoding a first polypeptide; A first intervening nucleic acid sequence encoding a first protein cleavage site, wherein the first protein cleavage site is an intein segment of the lon protease gene of pyrococcus or the klbA gene of pyrococcus or metanococus. Or by modified intein segments derived from them; A second nucleic acid sequence encoding a second polypeptide. Certain embodiments of the constructs and methods include the intein segment of the lon protease gene of Pyrococcus acci, Pyrococcus furiosus or Pyrococcus horicose OT3; Or intein segments of the klbA gene of Pyrococcus acci, pyrococcus puriosus or Metanococcus jannasquiy; Or use another intein segment.

Description

SORF CONSTRUCTS AND MULTIPLE GENE EXPRESSION

Before and after reference of related application

This application claims the benefit of U.S. Provisional Patent Application 61 / 256,544, filed October 30, 2009 by Gerald R. Carson et al., Which is incorporated by reference in its entirety.

Explanation of Research or Development Supported by the Federal Government

None

Sequence list, table or computer program list Compact  Reference to disk appendix

Not applicable (sequence list is provided but not as a compact disc supplement)

In the field of recombinant expression techniques, the achievement of high levels of production of desired protein products and the ability to produce products of the desired purity represents an ongoing challenge. While such challenges are particularly relevant for protein products, including biological therapeutic agents that are antibodies, progress in the art is also relevant for other biological agents. Certain aspects of the present invention focus at least in part on one or more aspects of this challenge.

The following abbreviations are applicable: ORF, open reading frame; sORF, single transcription decode frame; MW, molecular weight; HC or H, immunoglobulin heavy chains; LC or L, immunoglobulin light chains; Pab, Pyrococcus abyssi ); Pfu, Pyrococcus furiosus ); pho, Pyrococcus horikoshii ) OT3; aa or AA, amino acid (s); SP, single peptide; LCSP, light chain signal peptide; MTX, methotrexate.

Aspects of the present invention generally include expression cassettes for recombinant expression and processing including post-translational processing of recombinant polyproteins and pre-proteins. ), Vector constructs, recombinant host cells and methods. In aspects, the one or more expressed products is an immunoglobulin.

In aspects, the expression vector comprises one or more intein segments. In aspects, the intein segments originate from lon inteins of one or more of the organisms pyrococcus acci, pyrococcus puriosus, and pyrococcus horicose OT3.

In aspects, the architecture of the construct is constructed in terms of the order and presence or absence of particular components. In one embodiment, the order of the particular vector gene segments is HL, where H and L represent immunoglobulin heavy and light chains, respectively. In another embodiment, the order is LH. In a particular embodiment, the construct has a design labeled (-), where the negative signal is at the beginning of the ORF with the last amino acid of one signal peptide and intein and the first amino acid of the second protein subunit, for example, It has a methionine inserted between the mature antibody chains following the intein. In a particular embodiment, the construct has a design labeled with (+), wherein the plus signal is that of the first signal peptide at the beginning of the ORF and of the second signal peptide at the beginning of the second protein subunit of the intein. Indicates presence In a particular embodiment, the structure is HL (-).

In embodiments, the present invention will produce protein expression levels greater than 2, 5, 10, 20, 30, 40, or 50 micrograms per ml of secreted product when measured in culture supernatants from experiments under transient expression conditions. SORF (single transcription decoding frame) construct design. In aspects, the invention provides an sORF construct capable of producing protein expression levels greater than 20 micrograms per ml as measured in culture supernatants from experiments using conditions using a stable CHO (Chinese Hamster Ovary) cell expression system. To provide. In one embodiment, the expression level (μg / ml / day) ranges from 1 to 24, greater than 10 or greater than 20. In a particular embodiment, the expression level is 24 μg / ml / day. In embodiments, protein expression is a secreted antibody that self-assembles into multimeric units of heavy and light chains. In aspects, the antibody is IgG isotype.

In one aspect, the invention

(a) a signal peptide nucleic acid sequence encoding a signal peptide;

(b) a first nucleic acid sequence encoding a first polypeptide;

(c) a first intervening nucleic acid sequence encoding a first protein cleavage site, wherein the first protein cleavage site is a lon protease gene of pyrococcus or a klbA gene of pyrococcus or metanococus or , Modified intein segments derived therefrom); And

(d) providing an isolated or purified expression vector for producing one or more recombinant protein products comprising a single transcription translation frame insert comprising a second nucleic acid sequence encoding a second polypeptide, wherein A first intervening nucleic acid sequence encoding a first protein cleavage site is operatively located between the first nucleic acid sequence and the second nucleic acid sequence; Wherein the signal peptide nucleic acid sequence encoding the signal peptide is operably positioned before the first nucleic acid sequence; Wherein the expression vector may express a single transcription translation frame polypeptide cleavable at the first protein cleavage site.

For clarity in the context of embodiments involving various intervening segments and methods, intervening nucleic acid sequences encoding protein cleavage sites may be present such that the intervening nucleic acid sequences encode at least a first protein cleavage site. In circular intein, for example, the cleavage reaction is generally autonomous and proceeds in a rapid manner. Further explanation relies in part on understanding the underlying mechanism. From the post-processing point of view of the extein component, it can be understood that there is a first protein cleavage site and a second protein cleavage site towards each of the N-terminus and C-terminus of the intein segment. The design of the cleavage site is not intended to necessarily correspond to the order in which cleavage reactions may occur, and is single and relatively harmonized at one cleavage reaction site even when there is a recognition of dynamically apparent steps in the mechanism provided. It is recognized that there may be recognition of cleavage reactions as being a phenomenon. This technique also provides aspects of compositions and methods using intervening segments that include one or more cleavage sites, as would be understood in the art. Again, upon understanding of the processing mechanism, segments comprising one cleavage site or two cleavage sites may each allow partial or complete excision of the intervening segment.

In one embodiment, the intervening nucleic acid sequence also encodes a second protein cleavage site.

In one embodiment of the expression vector, the first protein cleavage site comprises an intein segment of the ion protease gene of Pyrococcus acci, Pyrococcus puriosus or Pyrococcus horicose OT3; Or intein segments of the klbA gene of Pyrococcus acci, Pyrococcus puriosus or Methanococcus jannaschii; Or by modified intein segments, each originating therefrom.

In one embodiment, the intein segment or modified intein segment encodes a penultimate residue at the end of lysine, serine but not histidine. In one embodiment, an intein segment or a modified intein segment may cleave the first polypeptide but not fully connect it to the second polypeptide.

In one embodiment, the first protein cleavage site is an intein segment comprising a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 4, 6, 7, 55, 35, 37, and 39 and derived therefrom. Provided by modified intein segments.

In one embodiment, the first polypeptide and the second polypeptide are capable of multimeric assembly. In one embodiment, at least one of the first polypeptide and the second polypeptide can secrete extracellularly. In one embodiment, at least one of the first polypeptide and the second polypeptide is of mammalian origin. In one embodiment, the first polypeptide comprises an immunoglobulin heavy chain or functional fragment thereof, and the second polypeptide comprises an immunoglobulin light chain or functional fragment thereof, wherein the first polypeptide is the second polypeptide At the top of the polypeptide of (5 'for the second polypeptide).

In an aspect of an expression vector, the vector comprises only one signal peptide nucleic acid sequence.

In one embodiment, the expression vector further comprises a third nucleic acid sequence encoding a third polypeptide, and a second intervening nucleic acid sequence encoding a second protein cleavage site; Here, the second intervening nucleic acid sequence and the third nucleic acid sequence are operatively located after the second nucleic acid sequence in this order.

In an embodiment of an expression vector, the first and second polypeptides are tumor necrosis factor-α, erythropoietin receptor, RSV, EL / selectin, interleukin-1, interleukin-12, interleukin-13, interleukin-17, interleukin. Antigen specificity directed against binding to an antigen selected from the group consisting of -18, interleukin-23, interleukin-33, CD81, CD19, IGF1, IGF2, EGFR, CXCL-13, GLP-1R, prostaglandin E2 and amyloid beta And functional antigen or other antigen recognition molecule.

In an embodiment of the invention, for the expression vector the first and second polypeptides comprise a pair of immunoglobulin chains from an antibody of D2E7, EL246, ABT-007, ABT-325, or ABT-874. In one embodiment, the first and second polypeptides are each independently from an immunoglobulin heavy chain or an immunoglobulin light chain segment from analog segments of D2E7, EL246, ABT-007, ABT-325, ABT-874, or other antibodies. Is selected.

In one embodiment, the expression vector further comprises a promoter regulatory component for said insert. In one embodiment, the promoter regulatory component is inducible or constitutive. In one embodiment, the promoter regulatory component is tissue specific. In one embodiment, the promoter comprises an adenovirus major late promoter.

In one aspect, the invention provides a host cell comprising the vector described herein. In one embodiment, the host cell is a prokaryotic cell. In one embodiment, the host cell is Escherichia coli. In one embodiment, the host cell is a eukaryotic cell. In one embodiment, the eukaryotic cells are selected from the group consisting of protozoal cells, animal cells, plant cells and fungal cells. In one embodiment, the eukaryotic cell is an animal cell selected from the group consisting of mammalian cells, avian cells and insect cells. In one embodiment, the host cell is a mammalian cell line. In one embodiment, the host cell is a CHO cell or a dihydrofolate reductase-deficient CHO cell. In one embodiment, the host cell is a HEK (human embryonic kidney) cell or an African green monkey kidney cell, eg, a COS cell. In one embodiment, the host cell is a yeast cell. In one embodiment, the yeast cells are Saccharomyces cerevisiae ). In one embodiment, the host cell is a Spodoptera frugiperda Sf9 insect cell.

In one embodiment, the present invention provides a method for producing recombinant polyprotein or a plurality of proteins, including culturing the host cell in culture medium under conditions sufficient to allow expression of the vector protein. In one embodiment, the method further comprises recovering and / or purifying the vector protein. In an aspect of the production method, multiple proteins are capable of multimeric assembly. In one embodiment, the recombinant polyprotein or multiple proteins are biologically functional and / or therapeutic.

In one embodiment, the present invention provides a method of producing a recombinant product comprising culturing a host cell under conditions sufficient to produce the recombinant product in a culture medium, wherein the product is an immunoglobulin protein or Functional fragments thereof, assembled antibodies, or other antigen recognition molecules. In one embodiment, the present invention provides a protein or polyprotein produced according to the methods described herein. In one aspect, the invention provides an immunoglobulin assembled according to the methods herein; Assembled other antigen recognition molecule; Or individual immunoglobulin chains or functional fragments thereof produced. In one embodiment, immunoglobulins; Other antigen recognition molecules; Or with respect to individual immunoglobulin chains or functional fragments thereof, tumor necrosis factor-α, erythropoietin receptor, RSV, EL / selectin, interleukin-1, interleukin-12, interleukin-13, interleukin 17, interleukin-18, Specific antigens that bind to interleukin-23, interleukin-33, CD81, CD19, IGF1, IGF2, EGFR, CXCL-13, GLP-1R, prostaglandin E2 or amyloid beta, where the antigen may be a ligand or a reverse receptor, etc. Ability to influence or contribute to In one embodiment, the immunoglobulin or functional fragment thereof is an immunoglobulin D2E7 or ABT-874 or the functional fragment is each a fragment thereof.

In one embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a protein and a pharmaceutically acceptable carrier.

In one aspect, the invention provides an expression vector described herein further comprising a nucleic acid sequence encoding a tag. In an embodiment of the vector construct, the intervening nucleic acid sequence further encodes a tag.

In one embodiment, the first and second polypeptides are tumor necrosis factor-α, erythropoietin receptor, RSV, EL / selectin, interleukin-1, interleukin-12, interleukin-13, interleukin 17, interleukin-18 , Functional with antigen specificity directed against binding to an antigen selected from the group consisting of interleukin-23, interleukin-33, CD81, CD19, IGF1, IGF2, EGFR, CXCL-13, GLP-1R, prostaglandin E2 and amyloid beta Antibodies or other antigen recognition molecules. In one embodiment, the first and second polypeptides comprise a pair of immunoglobulin chains from an antibody of D2E7, EL246, ABT-007, ABT-325, or ABT-874. In one embodiment, the first and second polypeptides are each independently from an immunoglobulin heavy chain or an immunoglobulin light chain segment from analog segments of D2E7, EL246, ABT-007, ABT-325, ABT-874, or other antibodies. Is selected.

In one embodiment, the vector further comprises a promoter regulatory component for said sORF insert. In one embodiment, the promoter regulatory component is inducible or constitutive. In one embodiment, the promoter regulatory component is tissue specific. In one embodiment, the promoter comprises an adenovirus major late promoter.

In one embodiment, the vector further comprises a nucleic acid encoding a protease capable of cleaving said first protein cleavage site. In one embodiment, the nucleic acid encoding a protease is operably located within the sORF insert; The expression vector further comprises an additional nucleic acid encoding a nucleic acid encoding a protease and a second cleavage site located between at least one of the first nucleic acid and the second nucleic acid.

In one aspect, the invention provides a host cell comprising the vector described herein. In one embodiment, the host cell is a prokaryotic cell. In one embodiment, the host cell is Escherichia coli . In one embodiment, the host cell is a eukaryotic cell. In one embodiment, the eukaryotic cell is selected from the group consisting of protozoal cells, animal cells, plant cells and fungal cells. In one embodiment, the eukaryotic cell is an animal cell selected from the group consisting of mammalian cells, avian cells and insect cells. In a preferred embodiment, the host cell is a CHO cell or a dihydrofolate reductase-deficient CHO cell. In one embodiment, the host cell is a COS cell. In one embodiment, the host cell is a yeast cell. In one embodiment, the yeast cell is Saccharomyces cerevisiae. In one embodiment, the host cell is an insect Spodoptera frugiperda Sf9 cell. In one embodiment, the host cell is a human embryonic kidney cell.

In one embodiment, the present invention provides a method for producing recombinant polyprotein or a plurality of proteins, including culturing the host cell under conditions sufficient to allow expression of the vector protein in the culture medium. In one embodiment, the method further comprises recovering and / or purifying the vector protein. In one embodiment, the plurality of proteins are capable of multimeric assembly. In one embodiment, the recombinant polyprotein or multiple proteins are biologically functional and / or therapeutic.

In one embodiment, the present invention provides a method of culturing a host cell according to claim 38 under conditions sufficient to produce an immunoglobulin protein or functional fragment thereof, assembled antibody, or other antigen recognition molecule in a culture medium. Including, immunoglobulin proteins or functional fragments thereof, assembled antibodies, or other antigen recognition molecules.

In one embodiment, the invention provides a protein or polyprotein produced according to the methods herein. In one aspect, the invention provides an assembled immunoglobulin produced according to the methods herein; Assembled other antigen recognition molecule; Or individual immunoglobulin chains or functional fragments thereof. In one embodiment, immunoglobulins; Other antigen recognition molecules; Or the individual immunoglobulin chains or functional fragments thereof have the ability to influence or contribute specific antigen binding to tumor necrosis factor-α, erythropoietin receptor, interleukin-18, EL / selectin or interleukin-12. . In one embodiment, the immunoglobulin is D2E7 or wherein the functional fragment is a fragment of D2E7.

In one embodiment, the present invention provides an expression vector, a host cell with the vector, a vector expression product, a pharmaceutical composition and / or a method of making or using any of the foregoing, wherein the vector is defined in the claims. A vector according to any one of claims 1 to 9 and further comprising a segment encoding a light chain signal peptide. In one embodiment, the encoded light chain signal peptide is a kappa light chain signal peptide selected from the group consisting of A17, A18, A19, A26, and H2G. In one embodiment, the encoded light chain signal peptide is VKII kappa light chain signal peptide A18, SEQ ID NO: 82 (amino acid sequence MRLPAQLLGLLMLWIPGSSA).

In one embodiment, the compositions of the present invention are isolated or purified.

In one embodiment, the composition of the present invention is a peptide compound. In one embodiment, the composition of the present invention is a nucleic acid compound. In one embodiment, the peptide compounds of the invention are assembled into a multimeric complex with a peptide or one or more other peptides.

In one embodiment, the present invention provides a pharmaceutical formulation comprising a composition of the present invention. In one embodiment, the present invention provides a method of synthesizing a composition of the present invention or a pharmaceutical formulation thereof. In one embodiment, the pharmaceutical formulation includes one or more excipients, carriers and / or other ingredients as would be understood in the art. In one embodiment, the effective amount of the composition of the present invention may be a therapeutically effective amount.

In one embodiment, the peptide composition of the present invention is prepared using recombinant methodology or synthetic techniques. In one embodiment, the nucleic acid compositions of the present invention are prepared using recombinant methodology or synthetic techniques.

In embodiments, the present invention provides a method of use in the manufacture of a medicament.

Other aspects, features, and advantages of aspects of the invention are apparent from the following description when considered in conjunction with the accompanying drawings and the teachings in the art.

In general, the terms and phrases used herein have their recognized meaning in the art as can be found by reference to standard textbooks, journal references, and content known to those skilled in the art. The definitions provided herein are intended to clarify their particular use in the context of aspects of the invention.

Without being bound by any particular theory, an understanding or certainty of the underlying principles or mechanisms associated with the present invention may be a discussion herein. Regardless of the ultimate accuracy of any description or hypothesis, aspects of the present invention may nevertheless be operational and useful.

1 shows a schematic of the sORF expression construct, pTT3 pab lon HL (−).
2 shows the structure of the sORF components in the expression construct for the D2E7 antibody.
3 shows the results of SDS-PAGE for protein analysis of sORF expression products. Secreted IgG molecules are purified by Protein A affinity chromatography and separated by SDS-PAGE under non-reducing (A) and reducing (B) conditions. Lanes and samples from left to right are as follows: (lane 1) MW reference marker; (2) control construct products; (3) Pab-lon mut A1; (4) Pab-lon mut A2; (5) pTT3 pfu lon YP, and (6) pTT3 pfu lon MA.
4 shows the results of SDS-PAGE for protein analysis of additional sORF expression products. Secreted IgG was purified by Protein A affinity chromatography and separated by SDS-PAGE under non-reducing (A) and reducing (B) conditions. Lanes and samples from left to right are as follows: (lane 1) MW marker; (2) control; (3) pTT3 pfu lon HL (−); And (4) pTT3 pfu lon MutA.
5 shows analysis of secreted antibodies produced from sORF constructs using the klbA intein. IgG products secreted from Pab-klbA HL (-) and Mja-klbA HL (-) constructs were purified by Protein A affinity chromatography and reduced (panels A, B, and C) and non-reduced (Panel D) It was separated by SDS-PAGE under the conditions. Panels A and D show images of stained gels; Panel B is an immunoblot using an antibody against human IgG1 Fc; Panel C is an immunoblot using antibodies against human kappa light chains. Lanes and samples from left to right are as follows: (lane 1) control; (2) Pab-klbA HL (-); And (3) Mja-klbA HL (-). The control is the same antibody produced from the expression of two separate transcription translation frames.
FIG. 6 shows the results of expression of a single transcriptional translation frame construct using Pab klbA intein with modifications to amino acid residues at the N-terminal splicing junction. Secreted IgG proteins were purified by Protein A affinity chromatography and separated under non-reducing and reducing conditions by SDS-PAGE. Lanes and samples from left to right are as follows: (lane 1) MW marker; (2) the same antibody produced using a conventional vector (control); (3) pTT3 Pab klba HL (−) wt; (4) pTT3 Pab klba HL (-) GC; And (5) pTT3 Pab klba HL (-) KC.
7 shows a schematic of the sORF expression construct, pA190-Pab-lon HL (−), adapted for use as a stable expression vector in the CHO cell line system.
8 shows the results of time and frequency of reaching culture consensus for a stable expression system of sORF construct transfection clone (sORF Pab lon construct).
9 shows a schematic of the structure for the sORF components of the transient expression construct with light chain linkage mutations. The series of constructs is designated "M1-X" (first line) and D2-X "(second week), where X represents any amino acid.
FIG. 10 shows IgG secretion results for a series of sORF constructs with light chain linkage mutations based on variations in Met1 residues.
11 shows IgG secretion results for a series of sORF constructs with light chain linkage mutations based on mutations in the Asp2 residue.
12 shows the results of SDS-PAGE analysis of protein products from examples of each of the Met1 and Asp2 series of constructs with light chain linkage mutations.
13 shows a schematic of the structure for the sORF components of a transient expression construct capable of expressing ABT-874 antibodies.
FIG. 14 shows hatched lines indicating the adjacent connections of segments H and F, relative to the end of the DOD endonuclease domain where the solvent is accessible for the introduction of an insert comprising a tag. The specific structural motif of the intein in relation to the position of the arrow).
15 shows a plasmid map of expression constructs with light chain signal peptide A18 for use in a transient transfection system of HEK293 cells.
16 shows the results of SDS-PAGE analysis of the product of antibody expression constructs.
17 shows the results of Western blot analysis of the product of antibody expression constructs from transfected cell lines comprising transiently infected HEK293 cells and stably transfected CHO cells.
18 shows a plasmid map of expression constructs using light chain signal peptide A18 for use in a stable transfection system of CHO cells.

DETAILED DESCRIPTION OF THE INVENTION

The invention can be further understood by the following non-limiting examples.

Certain information will be recognized by the description in the art, including those according to US 20070065912, issued March 22, 2007 by Carson et al.

The present invention provides systems, e.g., constructs and methods for the expression of biologically active protein or compound structures such as enzymes, hormones (e.g. insulin), cytokines, chemokines, receptors, antibodies or other molecules. to provide. Preferably, the protein is an interleukin, an immunoregulatory protein such as a full length immunoglobulin, a fragment thereof, another antigen recognition molecule as understood in the art, or other biotherapeutic molecule. An overview of this system is in the specific context of immunoglobulin molecules, where recombinant production is based on the expression of light and heavy chain coding sequences under the transcriptional control of a single promoter, where a single translational product (polyprotein) Conversion to separate heavy and light chains is mediated by intein components.

In one embodiment, the first or second chain of an immunoglobulin polyprotein molecule may be heavy or light chain. The sequence encoding the recombinant immunoglobulin segment may be a full length coding sequence or fragment thereof. In a specific embodiment, the second light chain coding sequence should be part of the sequence encoding the polyprotein to be processed in the practice of the present invention, ie there are three segments in any order comprising two light chains and one heavy chain. Consideration should be given. In a particular embodiment, the construct consists of these components and in the following order: a) IgH-IgL; b) IgL-IgH; c) IgH-IgL-IgL; d) IgL-IgH-IgL; e) IgL-IgL-IgH; f) IgH-IgH-IgL; g) IgH-IgL-IgH; And / or h) IgL-IgH-IgH. In one embodiment, the hyphen can indicate where the cleavage site sequence is located.

In addition, immunoglobulin heavy and light chain coding sequences are fused in frame to the intein coding sequence therebetween, wherein the intein is devoid of terminal or splicing activity of the designed heavy and light chains. This can be modified or naturally possible so that splicing preferably does not occur or splicing occurs with poor efficiency so that unspliced antibody molecules predominate. In addition, the modified inteins may still be further modified such that the endonuclease region (where the endonuclease region already exists) does not exist, provided site-specific proteolytic cleavage activity remains so that the light chain and Heavy chain antibody polypeptides are liberated from intervening intein portions of the main translation product. Either light or heavy chain antibody polypeptide can be N-extein and C-extein.

The vector may be any recombinant vector capable of expressing a full length polyprotein, such as an adeno-associated virus (AAV) vector, a lentiviral vector, a retroviral vector, a replica complement adenovirus vector, a replication deficient adenovirus Vector and gutless adenovirus vector, herpes virus vector or non-viral vector (plasmid) or any other vector known in the art, wherein the immunoglobulin or other protein (s) is expressed on a host cell. Choose the appropriate vector. Baculovirus vectors can be used for expression of genes in insect cells. Many vectors are known in the art and many are readily accessible in the art, either commercially available or otherwise.

Regulatory sequences, including promoters; Host cell

Vectors for recombinant immunoglobulin or other protein expression can include any of a number of promoters known in the art, wherein the promoter is constitutive, controllable or inducible, cell type specific, or tissue It is specific or species specific. Further specific examples include, for example, tetracycline-reactive promoters (Gossen M, Bujard H, Proc Natl Acad Sci US A. 1992, 15; 89 (12): 5547-51). Vector is a replicon (replicon) adjusted for the host cell, wherein the chimeric gene to be expressed, which is also a bacterial cell, and advantageously, a convenient cell for molecular biological manipulation, Escherichia coli (Escherichia coli ) in the presence of a replicon function.

The host cell for gene expression can be an animal cell, in particular a mammalian cell, but not limited to, or it can be a microbial cell (bacteria, yeast, fungus, but preferably eukaryotic) or plant cell. Particularly suitable host cells are Spodoptera Insect cultured cells, such as frugiperda ) cells, Saccharomyces yeast cells, such as cerevisiae or Pichia pastoris , Trichoderma fungi such as reesei , Aspergillus , Aureobasidum and Penicillium species, and CHO (Chinese hamster ovary), BHK (hamster cub kidney), COS, 293, 3T3 ( Various transgenic animal systems, including but not limited to mammalian cells, such as mice), Vero (African green monkey) cells, and pigs, mice, rats, sheep, goats, and cattle. Chicken systems for expression in egg white and transgenic sheep, goat and bovine systems are known for expression in milk, among others. Baculoviruses, in particular AcNPV vectors, can be used for expression of single ORF antibodies and cleavage of the invention using, for example, the expression of sORF under the regulatory control of polyhedrin promoters or other potent promoters in insect cell lines. And cell lines are well known in the art and commercially available. Promoters used in mammalian cells are constitutive (herpes virus TK promoter, see McKnight, cells 31: 355, 1982; SV40 early promoter, see Benoist et al. Nature 290: 304, 1981, Raus sarcoma Virus sarcoma virus promoter, Gorman et al. Proc. Natl. Acad. Sci. USA 79: 6777, 1982; cytomegalovirus promoter, Foecking et al. Gene 45: 101, 1980; mouse breast tumors Viral promoter, generally see Etcheverry in Protein Engineering: Principles and Practice, Cleland et al., Eds, pp. 162-181, Wiley & Sons, 1996) or may be regulated (metallothionein promoter, for example See, for example, Hamer et al. J. Molec. Appl. Genet. 1: 273, 1982). Vectors can be based on viruses that infect specific mammalian cells, in particular retroviruses, vaccinia and adenoviruses and derivatives thereof are known in the art and commercially available. Promoters include, but are not limited to, cytomegalovirus, adenovirus rate, and baccinia 7.5K promoter. Yeast and fungal vectors [see, eg, Van den Handel, C. et al. (1991) In: Bennett, JW and Lasure, LL (eds.), More Gene Manipulations in Fungi, Academy Press, Inc., New York, 397-428, and promoters are also well known and widely available. Enolase is a well known constitutive yeast promoter and alcohol dehydrogenase is a well known regulated promoter.

The selection of specific promoters, transcription termination sequences and any other sequences, such as sequences encoding tissue specific sequences, will largely be determined by the type of cell in which expression is desired. It may be a bacterial, yeast, fungal, mammalian, insect, chicken or other animal cell.

Signal  order

The coding sequence of the protein to be cleaved, proteolytically processed or self processed into a vector may further comprise one or more sequences encoding one or more signal sequences. These encoded signal sequences may be associated with one or more mature segments in the polyprotein. For example, the sequence encoding the immunoglobulin heavy chain leader sequence may precede the coding sequence for the heavy chain, in frame and operatively linked with the rest of the polyprotein coding sequence. Similarly, the light chain leader peptide coding sequence or other leader peptide coding sequence may comprise one or both of an immunoglobulin light chain coding sequence and a sequence encoding a protease recognition sequence or by an adjacent chain from a self-processing site (eg 2A). With the leader sequence-chain separated by, an appropriate read frame can be associated within the frame while maintaining an appropriate reading frame.

Stoichiometry of immunoglobulin heavy and light chains

In many of the embodiments herein, the immunoglobulin / antibody light chain (IgL) and heavy chain (IgH) are present at about a 1: 1 ratio (IgL: IgH) at the vector level or at the intracellular level expressed in the host cell. Meanwhile, recombinant attempts herein and in other documents rely on equimolar expression of heavy and light chains (see, eg, US Patent Publication No. 2005 / 0003482A1 or International Publication No. WO2004 / 113493). In an aspect, the invention provides methods and expression cassettes and vectors that co-express by chain self-processing or proteolytic processing when the main translation product is a polyprotein and uses a 2: 1 ratio of light and heavy chain coding sequences do. In embodiments, the ratio is greater than 1: 1, such as about 2: 1 or greater than 2: 1. In a particular embodiment, the light chain coding sequences are used in ratios greater than 1: 1 (IgL: IgH). In a specific embodiment, the ratio of IgL: IgH is 2: 1. Thus, in embodiments, the advantages provided by the sORF antibody expression technique include the ability to manipulate gene dose ratios for heavy and light chains, the proximity of heavy and light chain polypeptides for multi-subunit assembly in the ER, and high efficiency protein secretion. Include potential for

The present invention provides a sequence encoding a heavy chain and one or at least two light chains of an immunoglobulin (ie, an antibody); Further providing a stable clone of a host cell or host cell transformed or infected with a vector comprising a sequence encoding a cleavage site, such as a self-processing, protease recognition site or signal peptide present between them; It may further comprise a sequence or sequences encoding additional proteolytic cleavage sites. In addition, full-length recombination consisting of multiple subunits (eg, those that are naturally produced as two-chain or multi-chain molecules or pro-proteins and that are cleaved or processed to release precursor-derived proteins and active moieties) The use of such cells or clones in producing somatic immunoglobulins or fragments thereof or other biologically active proteins is within the scope of the present invention. Non-limiting examples include insulin, interleukin-18, interleukin-1, bone morphogenic protein 4, bone morphogenic protein 2, any other two clavicle morphogenic proteins, nerve growth factor, renin, chymotrypsin, transforming growth factor β , And interleukin 1β.

In a related aspect, the present invention provides recombinant immunoglobulin molecules or fragments thereof, or other proteins produced by such cells or clones, wherein the immunoglobulins are self-processing cleavage sites [eg, intein or hedgehook ( hedgehog) domains], cleavage sites or signal peptide cleavage and methods of producing the same, and amino acids derived from vectors and host cells. In aspects, the present invention provides a host cell containing one or more constructs as described herein.

The present invention provides single vector constructs for the expression of immunoglobulin molecules or fragments thereof and methods of using them in vitro or in vivo. The vector allows for expression of functional antibody molecules using a single promoter and transcript by having self-processing or other protease recognition sequences between the first and second immunoglobulin coding sequences and between the second and third immunoglobulin coding sequences. . Exemplary vector constructs include a sequence encoding a self-processing cleavage site between transcription translation frames and further adjacent to the self-processing cleavage site for removal of amino acids comprising the self-processing cleavage site following cleavage. Proteolytic cleavage sites. Vector constructs have been found to be useful in methods involving enhanced production of full length biologically active immunoglobulins or fragments thereof in vitro and in vivo. Other biologically active proteins having at least two different chains may be employed using the same strategy, although it is understood that the coding sequences of the chains may not be required to be present in more than one ratio relative to the coding sequences of the other chains. It can manufacture.

Although special compositions and methods are illustrated herein, it is understood that any of a number of alternative compositions and methods are applicable and suitable for use in practicing the present invention. It will also be appreciated that the evaluation of polyprotein expression cassettes and vectors, host cells and methods of the invention can be performed using process standards in the art. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombination techniques), microbiology, biochemistry and immunology, which are within the scope of those skilled in the art. Such techniques are described in Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989), each of which is hereby incorporated by reference; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir & C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller & M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., Eds., 1993); PCR: The Polymerase Chain Reaction, (Mullis et al., Eds., 1994); and Current Protocols in Immunology (J. E. Coligan et al., eds., 1991).

Unless otherwise indicated, all terms used herein have the same meaning as is understood by one of ordinary skill in the art, and the practice of the present invention refers to conventional techniques of microbiology and recombinant DNA technology, which are within the knowledge of those skilled in the art. will use it.

The term “modified,” as used herein generally in reference to a protein, refers to a segment in which at least one amino acid residue is substituted, deleted from, or added to the referenced molecule. Similarly, in the context of nucleic acids, the term refers to a segment in which at least one nucleic acid subunit has been substituted, deleted from, or added to a referenced molecule.

As used herein, the term “intein” refers to an internal segment of a protein that typically facilitates its removal and affects the ligation of the flanking segment known as extein. Many examples of inteins are recognized in various types of organisms, with some structural and / or functional features shared in some cases. The present invention can make extensive use of inteins, and variants thereof, that are recognized and additionally recognized or found. See, eg, Gogarten JP et al., 2002, Annu Rev Microbiol. 2002; 56: 263-87; Perler, F. B. (2002), InBase, the Intein Database. Nucleic Acids Res. 30, 383-384 (also via the Internet at the Web site of New England Biolabs, Inc., Ipswich, Mass .; http://www.neb.com /neb/inteins.html; Amitai G, et al., Mol Microbiol. 2003, 47 (1): 61-73; Gorbalenya AE, Nucleic Acids Res. 1998; 26 (7): 1741 -1748.Non-canonical inteins In a protein, an intein-containing unit or an intein splicing unit may be understood to include a portion of the flanking extein, wherein the structural phase may contribute to a reaction such as cleavage, ligation, etc. The term also refers to It may be understood as a category in referring to an intein-based system having a "modified intein" component.

As used herein, the term “modified intein” refers to an intein that is cleaved or cleaved by at least one amino acid residue substituted, deleted from, or added to an intein splicing unit. Synthetic intein or natural intein that is not fully linked.

As used herein, the term “vector” refers to a DNA or RNA molecule, such as a plasmid, virus or other vehicle, that contains one or more heterologous or recombinant DNA sequences and is designed for transfer between different host cells. The terms "expression vector" and "gene therapy vector" refer to any vector that is effective for incorporating and expressing heterologous DNA fragments in cells. The cloning or expression vector may comprise additional components, for example, the expression vector may have two replication systems so that it has two organisms, e.g., human cells for expression and prokaryotic hosts for cloning and amplification. It can be kept inside. Any suitable vector effective for introducing the nucleic acid into the cell can be used to allow protein or polypeptide expression to produce, for example, viral vectors or non-viral plasmid vectors. Any cell effective for expression, e.g., insect cells and eukaryotic cells such as yeast or mammalian cells, is also useful in practicing the present invention.

The terms “heterologous DNA” and “heterologous RNA” refer to nucleotides that are not endogenous (natural) to the cells or portions of the genome or vector in which they are present. In general, heterologous DNA or RNA is added to cells by induction, infection, transfection, transformation, electroporation, biolistic transformation, or the like. Such nucleotides generally comprise at least one coding sequence, but the coding sequence does not need to be expressed. The term “heterologous DNA” may mean “heterologous coding sequence” or “transgene”.

As used herein, the terms "protein" and "polypeptide" can be used interchangeably and are typically the desired "protein" and "polypeptide" expressed using the self processing cleavage site-containing vector of the present invention. Say. Such “proteins” and “polypeptides” can be any protein or polypeptide useful for investigational, diagnostic, or therapeutic purposes, as further described below. As used herein, polyprotein is a protein intended to be processed to produce two or more polypeptide products.

As used herein, the term "multimer" refers to a protein composed of two or more polypeptide chains (sometimes referred to as "subunits") that can be assembled to form a functional protein. Multimers may be composed of two (dimeric), three (trimeric), four (quameric) or aberrant (eg, meric, etc.) peptide chains. Multimers may be produced self-assembly or may require components such as catalysts to assist in assembly. The multimer may consist of the same peptide chain (alone-multimer) or only two or more different peptide chains (hetero-multimer). Such multimers may be structurally or chemically functional. Many multimers are known and used in the art, including but not limited to enzymes, hormones, antibodies, cytokines, chemokines and receptors. Thus, multimers can have both biological (eg, pharmaceutical) and industrial (eg, bioprocessing / bioproduction) uses.

As used herein, the term “tag” refers to a peptide that can be incorporated into an expression vector that can function to allow detection and / or purification of one or more expression products of a vector insert. Such tags are well known in the art and contain a biotinyl moiety that can be detected by the indicated avidin (eg, a strep containing fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Tavidin) to the polypeptide or may comprise a radiolabeled amino acid. Affinity tags such as FLAG, glutathione-S-transferase, maltose binding protein, cellulose-binding domain, thioredoxin, NusA, mystin, chitin-binding domain, cutinase, AGT, GFP, and others are used for protein expression and Widely used as in purification systems. Additional non-limiting examples of tags for polypeptides include, but are not limited to, histidine tags, radioisotopes or radionuclides (eg, ³ H, ¹⁴ C, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc). , ¹¹¹ In, ¹²⁵ I, ¹³¹ I, ¹⁷⁷ Lu, ¹⁶⁶ Ho, or ¹⁵³ Sm); Fluorescent tags (eg FITC, rhodamine, lanthanide phosphor), enzyme tags (eg horseradish peroxidase, luciferase, alkaline phosphatase); Chemiluminescent tags; Biotinyl group; Certain polypeptide epitopes recognized by a second reporter (eg, leucine zipper pair sequence, binding site for a second antibody, metal binding domain, epitope tag); And gadolinium chelates.

As used herein in connection with a viral gene therapy vector of the present invention, the term "replicating deficiency" means that the viral vector cannot independently replicate and package its genome independently. For example, if a cell of a subject is infected with rAAV virion, the heterologous gene is expressed in the infected cell, but the infected cell lacks the AAV rep and cap genes and the accessory function gene. Because of this, rAAV cannot replicate.

As used herein, "retroviral transfer vector" refers to an expression vector comprising a nucleotide sequence that encodes a transgene and further comprises a nucleotide sequence essential for packaging of the vector. Preferably, retroviral delivery vectors also include sequences essential for transgene expression in cells.

As used herein, "packaging system" refers to a set of viral constructs comprising genes encoding viral proteins included in packaging a recombinant virus. Typically, the constructs of the packaging system ultimately get incorporated into the packaging cells.

As used herein, a "second generation" lentiviral vector system refers to a lentiviral packaging system that lacks functional accessory genes such as from which accessory genes, vif, vpr, vpu, and nef are deleted or inactivated ( See, eg, Zufferey et al. 1997. Nat. Biotechnol. 15: 871-875.

As used herein, a "third generation" lentiviral vector system has the characteristics of a second generation vector system from which a lentiviral packaging system further lacking a functional tat gene such as a tat gene deleted or inactivated. Say Typically, the gene encoding rev is provided on a separate expression construct (see, eg, Dull et al. 1998. J. Virol. 72: 8463-8471).

As used herein in the context of a virus or viral vector, “pseudotyped” refers to the replacement of a native viral envelope protein with a heterologous or functionally modified viral envelope protein.

As used herein in reference to a recombinant DNA construct or vector, the term “operably linked” means that the nucleotide components of the recombinant DNA construct or vector are generally covalently linked to each other. In general, "operably linked" DNA sequences are contiguous and, in the case of a secretory leader, are contiguous and in the same reading frame. However, enhancers do not have to be contiguous with sequences whose expression is upregulated. The term is consistent with 'operably positioned'.

Enhancer sequences affect promoter-dependent gene expression and can be located within the 5 'or 3' region of a native gene. An "enhancer" is a cis-acting component that stimulates or inhibits transcription of adjacent genes. Enhancers that inhibit transcription are also termed "silencers". Enhancers may function in one orientation over the distance from the coding sequence and from the lower position of the transcribed region to several kilobase pairs (kb) (ie, associated with the coding sequence). In addition, an insulator or chromatin open sequence, such as a matrix attachment region (Chung, Cell, 1993, Aug 13; 74 (3): 505-14, Frisch et al, Genome Research, 2001, 12: 349- 354, Kim et al, J. Biotech 107, 2004, 95-105, can be used to enhance the transcription of stably integrated gene cassettes.

As used herein, the term “gene” or “coding gene” refers to a nucleic acid sequence that is transcribed (DNA) and translated (mRNA) into a polypeptide in vitro or in vivo when operably linked to an appropriate regulatory sequence. The gene is a region preceding and following a coding region, such as a 5 'untranslated (5' UTR) or "leader" sequence and a 3 'UTR or "trailer" sequence, and individual It may or may not include an intervening sequence (intron) between the coding segments (exons) of.

A "promoter" is a DNA sequence that promotes RNA synthesis by directing the binding of RNA polymerase, ie, a minimal sequence sufficient to direct transcription. Promoter and corresponding protein or polypeptide expression may be cell-type specific, tissue-specific or species specific. Also included in the nucleic acid constructs or vectors of the invention are enhancer sequences that may or may not be contiguous with the promoter sequence.

As used broadly herein, “transcriptional regulatory sequences” or expression control sequences often include physically related sequences and promoter sequences that modulate or regulate the transcription of related coding sequences in response to nutritional or environmental signals. Those related to the sequence can measure tissue or cell specific expression, response to environmental signals, binding of proteins to increase or decrease transcription, and the like. A “regulatory promoter” is any promoter whose activity is influenced by a factor that acts cis or trans (eg, an inducible promoter activated by an external signal or agent).

A “constitutive promoter” is in most cases a human CMV image that promotes the constitutive expression of cloned DNA inserts in any promoter, eg, mammalian cells, that directs RNA production in many or all tissue / cell types. Diet early enhancer / promoter region.

The terms "transcriptional regulatory protein", "transcriptional regulatory factor" and "transcriptional factor" are used interchangeably herein and refer to nuclear proteins that transcriptionally regulate the expression of a related gene or genes by binding to a DNA response component. Transcriptional regulatory proteins generally bind directly to the DNA response component, but in some cases binding to the DNA can ultimately bind to the DNA response component or indirectly as a means of binding to other proteins to which it is bound.

As used herein, "immunoglobulin" and "antibody" refer to Fa, F (ab ') ₂ , and Fv capable of binding a complete molecule and fragments thereof, eg, the desired antigenic determinant. These “immunoglobulins” and “antibodies” consist of two identical light chain polypeptide chains having a molecular weight of approximately 23,000 Daltons, and two identical heavy chains having a molecular weight of 53,000 to 70,000. The four chains are linked by disulfide bonds in the "Y" structure. Heavy chains are classified as gamma (IgG), mu (IgM), alpha (IgA), delta (IgD) or epsilon (IgE) and are the criteria for classifying immunoglobulins that determine the effector function of a given antibody. Light chains are classified as kappa or lambda. Where reference is made herein to "immunoglobulins or fragments thereof", such "fragments thereof" refer to immunologically functional immunoglobulin fragments, particularly their cognitive ligands having a binding affinity of at least 10% of complete immunoglobulins. It will be understood that the combination.

Fab fragments of antibodies are monovalent antigen-binding fragments of antibody molecules. Fv fragments are genetically engineered fragments containing the variable region of the heavy chain and the variable region of the light chain expressed as two chains.

The term “humanized antibody” refers to an antibody molecule in which one or more amino acids are replaced in a non-antigen binding region that is more similar to a human antibody while still retaining the original binding activity of the antibody (see US Pat. No. 6,602,503). number).

As used herein, the term “antigenic determinant” refers to a fragment of a molecule (ie epitope) in contact with a particular antibody. Multiple regions of a protein or peptides or glycopeptides of a protein or glycoprotein can induce the production of an antibody that specifically binds to a region or three-dimensional structure provided in the protein. These regions or structures are referred to as antigenic determinants or epitopes. Antigenic determinants can compete with a complete antigen (ie, an immunogen used to elicit an immune response) for binding to the antibody.

The term “fragment”, when referring to a recombinant protein or polypeptide of the invention, refers to an amino acid of a corresponding full length protein or polypeptide that retains at least one of the function or activity of the corresponding full length protein or polypeptide. A peptide or polypeptide having the same amino acid sequence as some but not all of the sequences. The fragment preferably comprises at least 20 to 100 contiguous amino acid residues of a full length protein or polypeptide.

As used herein, the term “administering” or “introducing” means delivering a protein (including immunoglobulin) to a person or animal in need thereof by any route known in the art. Pharmaceutical carriers and formulations or compositions are also known in the art. Routes of administration may include intravenous, intramuscular, intradermal, subcutaneous, transdermal, mucosal, intratumoral or mucosal. In addition, the terms may refer to delivering a vector for recombinant protein expression to a cell or cells in culture and to a cell or organ of a subject. Such administration or introduction may occur in vivo, in vitro or ex vivo. Vectors for expressing recombinant proteins or polypeptides typically include insertion of heterologous DNA into cells by physical means (eg, calcium phosphate transfection, electrophoresis, microinjection or lipid infection); Infections generally referring to introduction with an infectious agent, ie a virus; Or transfection, which typically refers to the transfer of genetic material from one microorganism with a viral agent (eg bacteriophage) to another or transduction, which means stable transfection of cells with a virus. Can be.

"Transformation" is commonly used to mean bacteria comprising heterologous DNA or cells that express an oncogene and thus have been converted into a continuous growth mode, eg, tumor cells. The vector used to “transform” the cell can be a plasmid, virus or other vehicle.

Typically, a cell is meant to be "transduced", "infected", "transfected" or "transformed" depending on the means used for administration, introduction or insertion of heterologous DNA (ie, vector) into the cell. do. The terms “transduced”, “transfected” and “transformed” can be used interchangeably herein regardless of the method of introducing heterologous DNA.

As used herein, the terms “stable transformed”, “stable transfected” and “genetransplantation” refer to cells having non-natural (heterologous) nucleic acid sequences integrated into the genome. Evidence of the establishment of a cell line or clone consisting of a population of daughter cells containing transfected DNA that reliably replicates by means of integration into the genome or as an episomal component. Is not stable, ie, transient In transient transfection, exogenous or heterologous DNA is expressed, but the introduced sequence is not integrated into the genome or the host cell cannot replicate.

As used herein, "ex vivo administration" refers to a subject in which a major cell is collected from a subject and the vector is administered to the cell to produce a transduced, infected or transfected recombinant cell and the same or different recombinant cells. Refers to the process to be re-administered.

"Multicistronic transcript" refers to an mRNA molecule containing one or more protein coding regions, or cistrons. MRNAs comprising two coding regions are referred to as "non-cistronic transcripts." A "5'-adjacent" coding region or cistron is a coding region whose transcription initiation codon (typically AUG) is closest to the 5 'end of the multicistronic mRNA molecule. A "5'-distal" coding region or cistron is one whose translational start codon (usually AUG) is not the closest start codon for the 5 'end of the mRNA.

The terms "5'-distant" and "lower" are used synonymously to refer to a coding region that is not close to the 5 'end of an mRNA molecule.

As used herein, “co-transcribed” means that two (or more) transcriptional translation frames or coding regions or polynucleotides are under transcriptional control of a single transcriptional control or regulatory component comprising a promoter.

As used herein, the term “host cell” refers to a cell that has been transduced, infected, transfected or transformed using a vector. Vectors can be plasmids, viral particles, phages and the like. Culture conditions such as temperature, pH and the like are those already used with the host cell selected for expression and will be apparent to those skilled in the art. It will be appreciated that the term "host cell" refers to cells that have been originally transduced, infected, transfected or transformed and their progenitor cells.

As used herein, the terms “biologically active” and “biologically active” refer to the activity that contributes to a particular protein in a cell line or cell-free system in culture, such as a ligand-receptor assay in an ELISA plate. "Biological activity" of an "immunoglobulin", "antibody" or fragment thereof refers to the ability to promote immunological function by binding to antigenic determinants. The "biological activity" of hormones or interleukins is known in the art.

As used herein, the terms “tumor” and “cancer” refer to cells that exhibit at least partial loss of control over normal growth and / or development. For example, often tumor or cancer cells generally have lossy contact inhibition and may be invasive and / or have the ability to metastasize.

Antibodies are immunoglobulin proteins that are heterodimers of heavy and light chains. Typical antibodies are multimers with two heavy chains and two light chains (or functional fragments thereof) associated together. Antibodies can often have additional polymer orders of structure that are dimeric, trimerous, tetrameric, meric, etc. that are dependent on isomorphism. They have proven extremely difficult to express in full length form from a single vector or two vectors in a mammalian culture expression system. Several methods are currently used for the production of antibodies: in vivo immunization of animals to produce “polyclonal” antibodies, in vitro cell culture of B-cell hybridomas to produce monoclonal antibodies (see: Kohler, et al. 1988. Eur. J. Immunol. 6: 511; Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988); And recombinant DNA techniques (see, eg, US Pat. No. 6,314,151 to Cabilly et al., Incorporated herein by reference).

The basic molecular structure of an immunoglobulin polypeptide is well known to include two identical light chains having a molecular weight of approximately 23,000 Daltons, and two identical heavy chains having a molecular weight of 53,000 to 70,000, wherein the four chains are of the “Y” structure. Connected by disulfide bonds. The amino acid sequence extends from the N-terminal end of the top of Y to the C-terminal end of the bottom of each chain. There is a variable region (about 100 amino acids in length) that provides specificity of antigen binding at the N-terminal end.

The present invention incorporates antigen-binding variable regions of both full length antibodies and antibody fragments with native sequences (ie, sequences produced in response to stimulation by antigen), both heavy and light chains within a single, stably folded polypeptide chain. Single-chain antibodies; Monovalent antibodies (which include heavy / light chain dimers that bind to the Fc region of a second heavy chain); "Which contains the complete" Y "region of the immunoglobulin molecule, ie the side chain of" Y "of the light chain or heavy chain alone, or a portion thereof (ie, one heavy chain and one light chain generally known as Fab '). Fab fragments; “Hybrid immunoglobulins” (eg, quadroma or bispecific antibodies as described in US Pat. No. 6,623,940) having specificity for at least two different antigens; “Complex immunoglobulins” that mimic heavy and light chains from different species or specificities; And some of the amino acid sequences of the heavy and light chains each are from one or more species (ie, the variable region is from one source, such as a murine antibody, while the fixed region is from another, such as a human antibody). ) And improved methods for the production of all types of immunoglobulins, including but not limited to "chimeric antibodies".

The compositions and methods of the present invention have been found to be useful in the production of immunoglobulins or fragments thereof, wherein the heavy or light chains are "mammals", "chimeras" or modified in ways that enhance their efficacy. Modified antibodies are both amino acid and nucleic acid sequence variants that retain the same biological activity in their unmodified form and are fixed regions that are modified to alter activity, i.e., complement complement, membrane interaction, and other effector functions. Or changes in variable regions that improve antigen binding properties. The compositions and methods of the present invention may further comprise catalytic immunoglobulins or fragments thereof.

A “variant” immunoglobulin-encoding polynucleotide sequence may encode a “variant” immunoglobulin amino acid sequence altered by one or more amino acids from a reference polypeptide sequence. This same discussion involved is applicable to other biologically active protein sequences (and their coding sequences) of interest. Variant polynucleotide sequences may encode variant amino acid sequences containing "conservative" substitutions, wherein the substituted amino acids have similar structural or chemical properties to the amino acids to which they are replaced. Variants of the protein of interest may be prepared using amino acid sequences that are substantially identical (at least about 80 to 99% identical, and all integers therebetween) with amino acid sequences of naturally occurring sequences, which are functionally equivalent, It forms a three-dimensional structure and retains the biological activity of naturally occurring proteins. It is well known in the biological arts that certain amino acid substitutions can be made within the protein sequence without affecting the function of the protein. In general, conservative amino acid substitutions or substitutions of similar amino acids are tolerated without affecting protein function. Similar amino acids can be similar in size and / or charge properties, for example aspartate and glutamate and isoleucine and valine are both pairs of similar amino acids. Substitution with one other is permitted unless the breakdown of the secondary secondary and tertiary structures is intended, unless intended. Similarity between amino acid pairs has been evaluated in the art in a number of ways. For example, Dayhoff et al., In Atlas of Protein Sequence and Structure, 1978. Volume 5, Supplement 3, Chapter 22, pages 345-352, incorporated herein by reference, can be used as a measure of amino acid similarity. Provides a frequency table of possible amino acid substitutions. Frequency tables by Dayhoff et al. Are based on comparisons of amino acid sequences for proteins having the same function from a variety of evolutionarily different sources.

Substitution mutations, insertions, and deletion variants of the described nucleotide (and amino acid) sequences can be readily prepared by methods well known in the art. These variants may be used in the same manner as specifically exemplified sequences so long as the variants have substantial sequence identity with the specifically exemplified sequences of the present invention and the desired functionality is preserved.

As used herein, substantial sequence identity refers to sufficient homology (or identity) to allow the variant polynucleotide or protein to function in the same capacity as the polynucleotide or protein from which the variant originates. Preferably, such sequence identity is greater than 70% or 80%, more preferably such identity is greater than 85% or such identity is greater than 90% and / or it is greater than 95% and 70 to 100 All integers between%. It is within the skill of the person skilled in the art that making substitution mutations, insertion mutations and deletion mutations that are equivalent in function or designed to improve sequence or other function provides methodological advantages. No aspect / variant that reads prior art items that can read or quantify any naturally occurring protein is intended to be within the scope of the invention as claimed. It is known in the art that certain obtained fragments and / or mutants of the original full length sequence may retain the desired characteristics of the full length sequence by causing the polynucleotide sequences of the present invention to be truncated and / or otherwise mutated. Well known in A wide variety of restriction enzymes suitable for generating fragments from larger nucleic acid molecules are well known. It is also well known that Bal31 exonuclease can be conveniently used for time-controlled limited digestion of DNA. See, eg, Maniatis et al. 1982. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, pages 135-139). See also Wei et al. 1983. J. Biol. Chem. 258: 13006-13512. By the use of Bal31 exonuclease (commonly referred to as the "erase-a-base" process), one of ordinary skill in the art would be able to remove the nucleotide from one or both ends of the subject nucleic acid to A broad spectrum of fragments can be produced that are functionally equivalent. One skilled in the art can generate hundreds of fragments of controlled varying lengths from position along the original coding sequence in this manner. One skilled in the art can routinely test or screen the resulting fragments for their characteristics and determine the utility of the fragments as taught herein. It is also well known that mutant sequences of full length sequences or fragments thereof can be readily produced by site directed mutagenesis. See, eg, Larionov, O.A., both of which are incorporated herein by reference. and Nikiforov, V.G. 1982. Genetika 18: 349-59; Shortle et al. (1981) Annu. Rev. Genet. 15: 265-94. Those skilled in the art will routinely produce deletion-, insertion-, or substitution-type mutations and the resulting mutants, such as hormones, cytokines, which contain the desired characteristics of full-length wild-type sequences, or fragments thereof. , Those with antigen-binding or other biological activity can be identified.

In addition, or alternatively, the variant polynucleotide sequence may encode a variant amino acid sequence containing "non-conservative" substitutions, wherein the substituted amino acids have different structural or chemical properties relative to the amino acid to which they are replaced. . Variant immunoglobulin-encoding polynucleotides may also encode variant amino acid sequences containing amino acid insertions or deletions, or both. In addition, the variant “immunoglobulin-encoding polynucleotide” may encode the same polypeptide as the reference polynucleotide sequence, but due to the degeneracy of the genetic code, one or more bases modified from the reference polynucleotide sequence have been modified. Has

The term "fragment", when referring to a recombinant immunoglobulin of the present invention, refers to a polypeptide having an amino acid sequence identical to, but not all of, the amino acid sequence of a corresponding full length immunoglobulin protein, which is corresponding full length Essentially retain the same biological function or activity as the protein of, or at least one of the corresponding full-length protein. The fragment preferably comprises at least 20 to 100 contiguous amino acid residues of a full length immunoglobulin and preferably retains the ability to bind the same antigen as the full length antibody.

As used herein, the term “sequence identity” refers to nucleic acid or amino acid sequence identity in two or more aligned sequences when aligned using a sequence alignment program. The term "% homology" is used interchangeably with the term "% identity" herein and when aligned using a sequence alignment program, the level of nucleic acid or amino acid sequence identity between two or more aligned sequences. Say For example, as used herein, 80% homology means the same as 80% sequence identity measured by an algorithm defined as understood in the art, and thus, homology of a given sequence is dependent upon the length of the provided sequence. Greater than 80% sequence identity over.

The optimal alignment of the comparative sequences is described, for example, by the local homology algorithm of Smith and Waterman. 1981. Adv. Appl. Math. 2: 482, Needleman and Wunsch. 1970. J By the homology alignment algorithm of Mol. Biol. 48: 443), these algorithms are investigated by the similarity method of Pearson and Lipman. 1988. Proc. Natl. Acad. Sci. USA 85: 2444. National Center for Biotechnology Information by Computerized Implementation of GAP, BESTFIT, FASTA, and TFASTA in the Genetics Computer Group of Madison, Wisconsin, the Wisconsin Genetics Software Package By the BLAST algorithm (see Altschul et al. 1990. J Mol. Biol. 215: 403-410) using publicly available software via the website (nlm.nih.gov/) or by visual observation (cf. Generally, by Ausubel et al., Supra) There. For the purposes of the present invention, optimal alignment of comparative sequences is described in Smith and Waterman. 1981. Adv. Appl. Math. 2: 482. In addition, Altschul et al. 1990 and Altschul et al. Most preferably by a local homology algorithm.

The term “identical” or “identity” percentages with respect to two or more nucleic acid or protein sequences is defined by the sequence comparison algorithm described herein, eg, the Smith-Waterman algorithm, others known in the art. Two or more sequences or subsequences having a defined percentage of identical or identical amino acid residues or nucleotides, when compared and aligned for maximum correspondence, as measured using, for example, BLAST, or visual observation. Say).

In accordance with the present invention, 80, 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% (and between 80 and 100 for percent values) relative to a native or reference sequence Also included are self-processing cleavage polypeptides having sequence identity of at least all integers) and sequence variants encoding the polypeptide itself. In addition, amino acid fragments of a polypeptide exhibiting contiguous elongation of at least 5, at least 10, or at least 15 units; And homologous fragments thereof according to the described identity conditions; And fragments of nucleic acid sequences exhibiting consecutive stretches of at least 15, at least 30, or at least 45 units. In a particular embodiment, the nucleic acid sequence or amino acid sequence is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or, for each sequence described herein 99.5% equal.

Nucleic acid sequences are considered to be "selectively hybridizable" to the reference nucleic acid sequence when the two sequences specifically hybridize to others under medium to high stringency hybridization and wash conditions. Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex or probe. For example, “maximum string display” typically occurs at about Tm-5 ° C. (less than 5 ° C. of the Tm of the probe); “High string display” occurs at about 5-10 ° C. below Tm; “Intermediate string display” occurs at about 10-20 ° C. below the Tm of the probe; “Low string display” occurs at about 20-25 ° C. below Tm. Functionally, maximum stringency conditions can be used to identify sequences with strict or near strict identity with hybridization probes; High stringency conditions are used to identify sequences that have at least about 80% sequence identity with the probe.

Medium and high stringency hybridization conditions are well known in the art (see, eg, Sambrook, et al, 1989, Chapters 9 and 11, and Ausubel, F. M., et al., 1993). Examples of high stringency conditions include hybridization at about 42 ° C. in 50% formamide, 5X SSC, 5X Denhardt's solution, 0.5% SDS and 100 μg / ml denatured carrier DNA followed by 2X SSC and 0.5 Two washes to room temperature in% SDS and an additional two washes in 0.1 X SSC and 0.5% SDS at 42 ° C. It is contemplated that within the scope of the invention are 2A sequence variants that encode polypeptides having the same biological activity as the naturally occurring proteins of interest and hybridize under medium to high stringency hybridization conditions.

As a result of the degeneracy of the genetic code, the same peptide sequence, including such structural components, self-processing components such as inteins, regulatory components such as signal peptidase cleavage sequences, or other components Multiple coding sequences can be produced that encode. For example, triplet CGT encodes the amino acid arginine. Arginine is alternatively encoded by the triplet nucleotide sequences CGA, CGC, CGG, AGA, and AGG. Thus, such substitutions of synonymous codons in the coding region are recognized to fall within the sequence variants encompassed by the present invention.

It is also recognized that such sequence variants may or may not hybridize to the parent sequence under high stringency conditions. This may be possible, for example, if the sequence variants contain different codons for each of the amino acids encoded by the parent nucleotides. Such variants are nevertheless specifically considered and included by the present invention.

The potential of antibodies as therapeutic modalities is currently limited by production capacity and the cost of current technology. Improved viral or non-viral single expression vectors for immunoglobulin (or other protein) production are capable of expressing and delivering two or more coding sequences, i.e., other proteins or immunoglobulins with dual- or multi-specificity from a single vector. To facilitate. The invention is further described herein in detail, including processed antibodies such as single chain antibodies, full length antibodies or antibody fragments, two chain hormones, two chain cytokines, two chain chemokines, two chain receptors, and the like. It is applicable to any immunoglobulin (ie, antibody) or fragment thereof or other multipart protein or binding protein pair as described.

Intaine

As used herein, intein is an expressed protein that is bound towards the N-terminus of the main expression product by N-extein and towards the C-terminus of the main expression product by C-extein. My segment is Naturally present inteins mediate ablation of intein and recombination of N- and C-exteins (protein linkages). However, with respect to the present expression product, the main sequence of the intein or flanking extein amino acid sequence is such that the cleavage of the protein backbone occurs in the absence or reduced or minimal amount of the linkage of the extein, thereby linking the extein protein to them. It is intended to be released from the main translation product (polyprotein) without forming a fusion protein. The intein region of the main expression product (the protein synthesized by mRNA, before any proteolytic cleavage) mediates proteolytic cleavage and intein / C-extein linkages in the N-extein / intein. In general, naturally occurring inteins also mediate splicing together with N-extein and C-extein (linking by the formation of peptide bonds). However, as applied to the goal of expressing two polypeptides in the present invention (as specifically illustrated by the heavy and light chains of the antibody molecule), it is preferred that no protein linkage occurs. This can be accomplished through mutations that do not have linking activity or by incorporating intein naturally. Alternatively, splicing can be prevented by mutations to change the amino acid (s) at or after the splicing site to prevent linkage of the released protein (Xu and Perler, 1996, EMBO). J. 15: 5146-5153). For example, Ser, Thr or Cys occurs normally at the beginning of C-extein and cannot be altered to modify or block splicing. In particular inteins, the effect on splicing prevents or reduces the ligation of expressed proteins.

Inteins are a class of proteins whose genes are found only in the genes of other proteins. Together with the flanking host genes named exteins, the intein is transcribed into a single mRNA and translated as a single polypeptide. Post-detoxification, the intein removes itself by initiating autocatalytic phenomena and connects the flanking host protein segments with new polypeptide bonds. The reaction is catalyzed solely by intein and does not require other cellular proteins, co-factors or ATP. Inteins are found in a variety of single cell organisms and they differ in size. Many inteins contain endonuclease domains that are counted for their mobility in the genome.

Intein mediated reactions have been used in biotechnology, in particular in vitro settings such as purification and protein chip construction, and in plant strain improvement. Perler, F.B. 2005. IUBMB Life 57 (7): 469-76. Mutations are introduced into native intein nucleotide sequences and some of these mutants have been reported to have altered properties (Xu and Perler, 1996. EMBO J. 15 (9), 5146-5153). In addition to the intein, ie the bacterial intein-like (BIL) domain and the hedgehog (Hog) self-processing domain, the other two members of the Hog / intein phase are also deciphered through similar mechanisms. It is known to catalyze post self-processing. See Dassa et.al. 2004. J. Biol. Chem. 279 (31): 32001-32007.

Inteins occur by in-frame insertion in specific host proteins. In a self-splicing reaction, the intein is self-resected from the precursor protein, while the flanking region, the extein, is linked to restore host gene function. These components also contain endonuclease functions that are counted for their mobility in the genome. Inteins occur at various sizes (134-1650 amino acids) and they have been identified in the genomes of eubacteria, eukaryota and arcaea. Experiments using the model splicing / reporter system have shown that endonucleases, protein cleavage, and protein splicing functions can be isolated (Xu and Perler. 1996. EMBO J. 15: 5146-5153 ). The examples described below include Pyrococcus horikoshii ) Pho Pol I, Saccharomyces cerevisiae ) VMA, and Synechocystis spp . Inteins from) are used to generate fusion proteins with sequences from antibody heavy and light chains. Mutations in intein designed to eliminate the splicing ability of intein result in single-chain polypeptides that self-cleavage to produce correctly encoded antibody heavy and light chains. This strategy can be used similarly for the expression of other multichain proteins, hormones or cytokines, which can also be tailored for the processing of precursor proteins (proproteins) for their mature, biologically active forms. Pyrococcus horikoshii Pho Pol I, S. The use of VMA, and cinechocytis subspecies inteins in cerevisiae is specifically illustrated herein, but other inteins known in the art can be used in the polyprotein expression vectors and methods of the present invention.

Pyrococcus horikoshii Pho Pol I, S. Many other inteins are known in the art in addition to VMA in cerevisiae, and cinechosis subspecies intein. See, eg, Perler, F.B. 2002, InBase, the Intein Database, Nucl. Acids Res. 30 (1): 383-384 and the Intein Database and Registry, available via the New England Biolabs website, e.g., at http://tools.neb.com/inbase/]. Inteins have been identified in a wide range of organisms such as yeast, mycobacteria and extremely thermophilic archaebacteria. Certain intaines have endonuclease activity and site-specific protein cleavage and splicing activity. Endonuclease activity is not essential to the practice of the present invention; The endonuclease coding region may be deleted provided that the proteolytic activity is maintained.

The mechanism of the protein splicing process has been studied in great detail (Chong et al. 1996. J. Biol. Chem. 271: 22159-22168; Xu and Perler. 1996. EMBO J 15: 5146-5153). Amino acids have been found at the intein and extein splicing sites (Xu et al. 1994. EMBO J 13: 5517-5522). Certain constructs described herein contain an intein sequence fused at the 3′-end of the first coding sequence and a second coding sequence fused in-frame at the C-terminus of the intein. Suitable intein sequences can be selected from any of the proteins known to contain protein splicing components. Databases containing all known inteins can be found from the World Wide Web (Perler, F. B. 1999. Nucl. Acids Res. 27: 346-347). The intein coding sequence is fused (in-frame) at the 3 'end to the 5' end of the second coding sequence. For targeting of the protein to a particular organ, the appropriate peptide signal can be fused to the coding sequence of the protein.

After the second extein coding sequence, the intein coding sequence-extein coding sequence can be repeated as often as desired for the expression of multiple proteins in the same cell. For multi-intein containing constructs, it may be useful to use intein components from different sources. After the sequence of the final gene is expressed, it is preferably inserted comprising a transcription terminal sequence and advantageously a polyadenylation sequence. The order of the polyadenylation sequence and the terminal sequence can be as understood in the art. In an aspect, the polyadenylation sequence may precede the terminal sequence.

Modified intein splicing units are designed such that this desired modified intein can catalyze the ablation of extein from the intein but not catalyze the linkage of the extein (see, eg, US Patent no. 6,526,6 and US Patent Publication No. 20020129400. Mutagenesis of the C-terminal extein linkage in Pyrococcus spp. GB-D DNA polymerase produced an altered splicing component that induces cleavage of the extein but prevents subsequent linkage of the extein (see Xu and Perler. 1996. EMBO J 15: 5146-5153). Mutations of aserine or glycine (from Ser to Ala or Gly) of serine 538 induced cleavage but prevented linkage. Ser to Met or Ser to Thr at these positions are also used to achieve expression of polyproteins that are cleaved into separate segments and are not at least partially re-linked. Mutations of equivalent residues in other intein splicing units can also prevent linkage of the extein segment due to the relative conservation of amino acids at the C-terminal extein linkage to the intein. In the example of low conservative / homologous, for example, an extein segment provided with a systemic alteration of the residues of the first few, for example about 5 C-exteins, and / or the last few residues of the intein segment In particular, for the ability to support non-splicing of the extein segments as described herein and as understood in the art. There is an intein that does not contain an endonuclease domain; These are the Synechocystis spp dnaE intein and Mycobacterium xenopi GyrA proteins (Magnasco et al, Biochemistry, 2004, 43, 10265-10276; Telenti et al. 1997. Bacteriol. 179: 6378-6382). Others have been found in nature or have been artificially produced by removing endonuclease coding domains from sequences encoding endonuclease-containing inteins (Chong et al. 1997. J. Biol. Chem. 272: 15587-15590. If desired, the intein was originally selected to consist of the minimum number of amino acids required to perform splicing functions such as intein from the Mycobacterium genophyte GyrA protein (Telenti et al. 1997. See above). In an alternative embodiment, phosphorus without endonuclease activity, such as intein from Mycobacterium genophyte GyrA protein modified to remove endonuclease domain or VMA intein to Saccharomyces cerevisiae Tayne is selected (Chong et al. 1997. see above).

Further modifications of the intein splicing unit can cause the protein dose to be controlled by simply modifying the gene sequence of the splicing unit by causing the reaction rate of the cleavage reaction to be altered.

In one embodiment, the first residue of the C-terminal extein is processed to contain glycine or alanine, a modification that has been found to prevent extein linkage using pyrococcus species GB-D DNA polymerase (see Xu and Perler. 1996. EMBO J 15: 5146-5153). In this embodiment, the preferred C-terminal extein protein naturally contains a glycine or alanine residue followed by an N-terminal methionine in its natural amino acid sequence. Fusion of the extein glycine or alanine to the C-terminus of the intein provides a natural amino acid sequence after processing of the polyprotein. In other embodiments, artificial glycine or alanine is located in the C-terminal extein by altering the native sequence or adding additional amino acid residues to the N-terminus of the native sequence. In this embodiment, the natural amino acid sequence of the protein will be altered by one amino acid after polyprotein processing. In a further aspect, other variations useful in the present invention are described in US 7026526.

In one embodiment, the intein is according to the same as in US Pat. No. 7,026,526. In a particular embodiment, the intein is Pyrococcus spp. GB-D DNA polymerase intein. In one embodiment, the mutation of the C-terminal extein serine to alanine or glycine forms a modified intein splicing component that can promote excision of the polyprotein but does not promote linkage of the extein units. In one embodiment, the intein is the Mycobacterium genophyte GyrA minimal intein of US Pat. No. 7,026,526. In one embodiment, the mutation of the C-terminal extein threonine to alanine or glycine forms a modified intein splicing component that promotes ablation of the polyprotein but does not link the extein units.

It will be appreciated that for certain inteins as described herein, embodiments of the constructs and methods can produce improved expression of protein products secreted against multimeric proteins, particularly including antibodies.

Signal Peptide  And Signal Peptidase

Signal hypotheses in which proteins contain information in their amino acid sequence for protein targeting to the membrane have been known for more than 30 years. Milstein and co-researchers found that the light chain of IgG from melanoma cells was synthesized in high molecular weight form and converted to its mature form when cytoplasmic reticulum vesicles (microsomal bodies) were added to the detoxification system. A model based on these results containing a protease that converts the precursor protein form to mature form by removing the amino-terminal extension peptide was proposed. The signal hypothesis soon expanded to contain distinct targeting signals in proteins localized to different intracellular membranes such as mitochondria and chloroplasts. These apparent targeting sequences were later found to be cleaved from the protein exported by specific signal peptidase (SPase).

There are at least three distinct SPases involved in cleaving signal peptides in bacteria. SPase I can process non-lipoprotein substrates exported by the SecYEG pathway or the twin arginine translocation (Tat) pathway. Lipoproteins released by the Sec pathway are cleaved by SPase II. SPase IV cleaves prepilin-like proteins that are components of type IV prepilins and type II secretion devices.

In eukaryotic cells, proteins targeted to reticulum (ER) membranes are mediated by signal peptides that target proteins either co-detoxically or post-detoxically against the Sec61 translocation machinery. ER signal peptides have characteristics similar to those of their bacterial counterparts. The ER signal peptide is cleaved from the outflowed protein after it is flowed into the ER lumen by the signal peptidase complex (SPC).

Signal peptides that classify proteins at different positions in eukaryotic cells should be distinguished because these cells contain many different membranes and aqueous compartments. Proteins targeted to ER often contain cleavable signal sequences. Surprisingly, many artificial peptides can function as translocation signals. The most important key feature is believed to be hydrophobicity exceeding this particular limit. ER signal peptides have a higher content of leucine residues than bacterial signal peptides. Signal recognition particles (SRP) bind to cleavable signal peptides after they are generated from ribosomes. SRP is required to target adjacent proteins against the ER membrane. After translocation of the protein into the ER lumen, the spilled protein is processed by SPC. Another embodiment takes advantage of signal (leader) peptide processing enzymes occurring naturally in eukaryotic cells.

Most known ER signal peptides are either N-terminal cleavable or not internally cleavable. Recently, many viral polyproteins, such as those found in hepatitis C virus, hantavirus, flavivirus, rubella virus, and influenza C virus, have been identified by ER SPC. It has been found to contain internal signal peptides that are most frequently cleaved. These studies in the maturation of polyproteins indicate that SPC can cleave not only after amino-terminal located signal peptides but also after internal signal peptides. Thus, mutagenesis of the predicted signal peptidase substrate specific components can block viral infectivity.

Presenilin-type aspartic protease signal peptide peptidase (SPP) cleaves signal peptides within their transmembrane region. SPP is essential for the generation of signal peptide-derived HLA-E epitopes in humans.

Signal peptidase is well known in the art. See, eg, Paetzel M. 2002. Chem. Rev. 102 (12): 4549; Pekosz A. 1998. Proc. Natl. Acad. Sci. USA. 95: 13233-13238; Marius K. 2002. Molecular Cell 10: 735-744; Okamoto K. 2004. J. Virol. 78: 6370-6380, Vol. 78; Martoglio B. 2003. Human Molecular Genetics 12: R201-R206; And Xia W. 2003. J. Cell Sci. 116: 2839-2844.

Embodiments of the invention use an internal cleavable signal peptide for expression of a polypeptide in a single transcript. The single transcribed polypeptide is then cleaved by SPC, leaving the individual peptides separate or assembling the individual peptides into proteins. The method of the present invention is applicable to the expression of immunoglobulin heavy and light chains in a single transcribed polypeptide and is assembled into mature immunoglobulins after cleavage. The technique is applicable to polypeptides cytokines, growth factors, or various other proteins such as IL-12p40 and IL-12p35 in a single transcribed polypeptide, followed by IL-12, or a single transcribed poly. Assembled with IL-12p40 and IL-23p19 in the peptide and then with IL-23.

Signal peptidase trials are applicable to mammalian expression vectors that produce expression of functional antibodies or other processed products from precursors or polyproteins. In the case of an antibody, it is produced from a vector as a polyprotein containing both heavy and light chains with an intervening sequence between the heavy and light chains, an internal cleavable signal peptide. The internally cleavable signal peptide can be cleaved by an ER-residue protease, mainly a signal peptidase, a presenilin or a presenilin-like protease, leaving the heavy and light chains folded and assembled to produce a functional molecule, Preferably it is secreted. In addition to internal cleavable signal peptides derived from hepatitis C virus, other internal cleavable sequences that can be cleaved by the ER-residue protease can also be substituted. Similarly, the practice of the present invention need not be limited to host cells where the signal peptidase affects cleavage, but also includes presenilins, presenilin-like proteases, and other proteases for processing polypeptides. One includes, but is not limited to, proteases. These proteases have been considered in the cited literature among others.

In addition, the present invention is not limited to the expression of immunoglobulin heavy and light chains, but it also includes other polypeptides and polyproteins that are expressed as single transcripts and then release each individual peptide or protein by internal signal peptide cleavage. These proteins may or may not be assembled together in mature products.

In addition, the individual polypeptides are expression constructs present in an alternative sequence, ie, "peptide 1 -internally cleavable signal peptide-peptide 2" or "peptide 2-inner cleavable signal peptide-peptide 1". The invention also encompasses the expression of two or more peptides linked by an internal cleavable signal peptide such as “peptide 1-internal cleavable signal peptide-peptide 2-internal cleavable signal peptide-peptide 3” and the like.

The invention also applies to other protease cleavage sites in connection with expression and expression constructs of both type I and type II transmembrane proteins.

The invention may also use internal cleavable signal peptides for maturation of one or more polypeptides in a polyprotein encoded within a single transcript. Thereafter, the single transcribed polypeptide is cleaved by SPC, leaving individual peptides assembled into separate individual peptides or proteins. Aspects of the present invention include compositions and methods for expressing immunoglobulin heavy and light chains in a single transcribed polypeptide, ultimately with assembly into mature immunoglobulins. The present invention provides for the expression of a polypeptide cytokine, growth factor, or a variety of other proteins, for example, IL-12p40 and IL-12p35 in a single transcribed polypeptide, followed by IL-12, or a single Applicable for assembly with IL-12p40 and IL-23p19 in the transcribed polypeptide followed by assembly with IL-23.

Modification of Signal Peptides

In an embodiment of the sORF construct, a modified signal peptide is used. For example, for the construction of heavy-int-light chains, antibody secretion concentrations increased about 10-fold when the hydrophobicity of the light chain signal peptide sequences was reduced through site-directed mutagenesis. The signal peptide can be used as described in US Patent Application Publication No. US 20070065912, issued March 22, 2007 to Carson et al.

tag

Aspects of the sORF construct design of the present invention include the use of modified inteins containing a tag, preferably an inner tag. Various tags are known in the art. Tags of the present invention include, but are not limited to, fluorescent tags and chemiluminescent tags. Using this construct, the amount of polyprotein expressed can be monitored using fluorescence detection in individual cells. In addition, these cells can be sorted according to protein expression levels using fluorescent activated cell sorting (FACS). The use of such a tag is particularly useful for the production of stable cell lines since it allows the selection of high producing cells or cell lines via FACS analysis. As taught herein, full length inteins have been observed in cell lysates after their self-cleavage from flanking antibody heavy and light chains. This provides bases for the detection of fluorescently labeled intaines and their use in the production of stable cell lines. The tag can also be used in the purification of proteins.

mini- Intaine

blood. Horikosii PolI Intein and Sce. Because the endonuclease regions present in many inteins, including the VMA intein, are not particularly advantageous in the case of gene expression systems, the endonuclease domains are optionally deleted and replaced with bovine linkers to replace the "mini-inteins". Can be generated. These engineered mini-intaines are also useful for designing the described constructs, which have significantly smaller intein coding regions, allowing for larger sequences encoding the desired polypeptide and / or larger size of recombinant DNA molecules. It offers the advantage of allowing ease of handling.

In embodiments, it is advantageous to use an antibody or analog thereof having complete human properties. These reagents avoid undesirable immune responses induced by antibodies or analogs derived from non-human species. To focus on possible host immune responses to amino acid residues derived from self-processed peptides, the coding sequence for the proteolytic cleavage site is inserted between the coding sequence for the first protein and the coding sequence for the self-processed peptide. Insertion (using standard methodologies known in the art) can be inserted to remove self-processing peptide sequences from expressed polypeptides, ie antibodies. This finds particular utility in therapeutic or diagnostic antibodies for use in vivo.

Vectors containing gene transfer and viral vectors

The present invention contemplates the use of any of a variety of vectors for the introduction of constructs comprising coding sequences for two or more polypeptides or proteins and cleavage sequences that are self processed into cells. Many examples of gene expression vectors are known in the art and may be of viral or non-viral origin. Non-viral gene delivery methods that can be used in the practice of the present invention include, but are not limited to, plasmids, liposomes, nucleic acid / liposomal complexes, cationic lipids, and the like.

Viruses and other vectors can efficiently transduce cells and introduce their own DNA into host cells. In generating recombinant viral vectors, non-essential genes are replaced with expressible sequences encoding the protein or polypeptide of interest. Exemplary vectors include viral and non-viral vectors, such as adenovirus (Ad) vectors, including retroviral vectors (including lentiviral vectors), replication complements, lack of replication, and gutless forms, Adeno-associated virus (AAV) vector, Simian virus 40 (SV-40) vector, bovine papilloma vector, Epstein-Barr vector, herpes vector, bacsinia vector, molony murine leukemia vector, harvey Harvey murine sarcoma virus vectors, murine breast tumor virus vectors, Rous sarcoma virus vectors, and non-viral plasmids. Baculovirus vectors are well known and suitable for expression in insect cells. Many vectors suitable for expression in mammalian or other eukaryotic cells are well known in the art and many are commercially available. Commercially available sources include Stratagene (La Jolla, Calif.); Invitrogen (Carlsbad, CA); Promega (Madison, WI) and Sigma-Aldrich (St. Louis, MO). Many vector sequences are available through GenBank, and additional information about the vector is available on the Internet through the Riken BioSource Center.

In one aspect, the vector typically comprises a replication origin and the vector may or may not also include a "marker" or "selectable marker" function by which the vector can be identified and selected. While any selectable marker can be used, selectable markers for use in recombinant vectors are generally known in the art and the selection of the appropriate selectable marker will depend on the host cell. Examples of selectable marker genes that encode proteins that confer resistance to antibiotics or other toxins are ampicillin, methotrexate, tetracycline, neomycin (Southern et al. 1982. J Mol Appl Genet. 1: 327-41) , Mycophenolic acid (Mulligan et al. 1980. Science 209: 1422-7), puromycin, zeomycin, hygromycin (Sugden et al. 1985. Mol Cell Biol. 5: 410-3), Dihydrofolate reductase, glutamine synthetase, and G418. As will be appreciated by those skilled in the art, expression vectors are typically appropriate for the coding sequence (s) to be expressed, such as replication origin, promoter operably linked to the coding sequence or sequences to be expressed, and ribosomal binding sites, RNA stubs. Fliesing site, polyadenylation site, and transcription terminator sequence.

References to vectors or other DNA sequences as “recombinants” merely recognize operable linkage of DNA sequences that have been isolated or found from nature and are typically not operably linked. Regulatory (expression and / or control) sequences are operably linked to nucleic acid coding sequences when the expression and / or control sequences control transcription and, optionally, the translation of nucleic acid sequences. Thus, expression and / or control sequences may include promoters, enhancers, transcription terminators, start codons (ie ATG) 5 ′ for the coding sequence, splicing signals for introns, and stop codons.

Adenovirus gene therapy vectors are known to exhibit strong transient expression, excellent titers, and the ability to transduce dividing and non-dividing cells in vivo (Hitt et al. 2000. Adv in Virus Res 55). 479-505). Recombinant Ad vectors include a packaging site capable of introducing the vector into a replication-deficient Ad virion; Coding sequences for two or more polypeptides or proteins of interest, such as the heavy and light chains of an immunoglobulin of interest; And a sequence encoding a cleavage site that is self-processed alone or in combination with additional proteolytic cleavage sites. Other components essential or helpful for introduction into infectious virions include 5 'and 3' Ad ITRs, E2 genes, regions of E4 genes and optionally E3 genes.

Replication-deficient Ad virions encapsulating recombinant Ad vectors are prepared using Ad packaging cells and packaging techniques by standard techniques known in the art. Examples of these methods can be found, for example, in US Pat. No. 5,872,005. Coding sequences for two or more desired polypeptides or proteins are generally inserted into the adenovirus within the deleted E3 region of the viral genome. Preferred adenovirus vectors for use in practicing the present invention do not express one or more wild type Ad gene products, such as E1a, E1b, E2, E3, and E4. Preferred embodiments are virions typically used with packaging cell lines that complement the functions of E1, E2A, E4 and optionally E3 gene regions (see, eg, US Pat. Nos. 5,872,005, 5,994,106, 6,133,028 and No. 6,127,175).

As used herein, “adenovirus” and “adenovirus particles” include all serotypes and subtypes, and both naturally occurring and recombinant forms, except where otherwise indicated and are the virus itself or derivatives thereof. Such adenoviruses may be wild type or may be modified in various ways as known in the art or as discussed herein. Such modifications include modifications to the adenovirus genome that are packaged into particles to produce infectious viruses. Such modifications include deletions known in the art, such as one or more of one or more El, Elb, E2a, E2b, E3, or E4 coding regions. Exemplary packaging and producer cells are from 293, A549 or HeLa cells. Adenovirus vectors are purified and formulated using standard techniques known in the art.

Adeno-associated virus (AAV) is a helper-dependent human parvovirus that can potentially infect cells by chromosomal integration. Because of its ability to integrate chromosomes and its nonpathogenic nature, AAV is significantly effective as a human gene therapy vector. For use in practicing the present invention, rAAV virions can be produced using standard methodologies known to those skilled in the art, which are components that are operably linked in the transcription direction, including transcription initiation and termination sequences, and desired coding sequences. It is constructed to include a control sequence comprising the (s). More specifically, the recombinant AAV vectors of the present invention may comprise a packaging site that allows the vector to be incorporated into a replication-deficient AAV virion; Coding sequences for two or more polypeptides or proteins of interest, such as the heavy and light chains of an immunoglobulin of interest; A sequence encoding a cleavage site that is self-processed alone or in combination with one or more additional proteolytic cleavage sites. AAV vectors for use in practicing the present invention include control sequences comprising transcription initiation and termination sequences as components in which they are also operably linked in the transcription direction. These components are flanked at the 5 'and 3' ends by functional AAV ITR sequences. By "functional AAV ITR sequence" is meant that the ITR sequence functions as intended for the rescue, replication, and packaging of AAV virions.

Recombinant AAV vectors are also characterized in that they can direct the expression and production of selected recombinant polypeptides or protein products in target cells. Thus, the recombinant vector comprises at least both the sequence of AAV essential for encapsidation and the physical structure for infection of the recombinant AAV (rAAV) virion. Thus, AAV ITRs for use in expression vectors need not have a wild type nucleotide sequence (see, eg, Kotin. 1994. Hum. Gene Ther. 5: 793-801), and are not required for insertion, deletion or substitution of nucleotides. Or AAV ITRs may originate from any of several AAV serotypes. In general, the AAV vector can be any vector derived from adeno-associated virus serotypes known in the art.

Typically, an AAV expression vector is introduced into a producer cell, followed by the introduction of an AAV helper construct, wherein the helper construct is provided with an AAV coding region that can be expressed in the producer cell and complements the AAV helper function absent in the AAV vector. Include. Helper constructs down-regulate the expression of the huge Rep proteins (Rep78 and Rep68) by mutating the start codon with p5, typically from ATG to ACG, as described in US Pat. No. 6,548,286, which is incorporated herein by reference. It can be designed to adjust. This involves the introduction of helper viruses and / or additional vectors into the producer cells, where the helper viruses and / or additional vectors provide an auxiliary function that can support efficient rAAV virus production. Thereafter, producer cells are cultured to produce rAAV. These steps are performed by standard methodology. Replication-deficient AAV virions capable of encapsulating recombinant AAV vectors of the invention are prepared using AAV packaging cells and packaging techniques by standard techniques known in the art. Examples of these methods are described in U. S. Patents 5,436, 146, which is incorporated by reference in its entirety; 5,753,500, 6,040,183, 6,093,570 and 6,548,286. Additional compositions and methods for packaging are also described in US Pat. Pub. No. 2002/0168342 to Wang et al., Incorporated herein by reference in its entirety, and includes techniques that are within the knowledge of one of ordinary skill in the art. .

In practicing the present invention, host cells for producing rAAV or other vector expression vector virions include mammalian cells, insect cells, microorganisms and yeast. The host cell may also be a packaging cell in which the AAV (or other) rep and cap genes remain stable in the host cell or producer cell in which the AAV vector genome is stably maintained and packaged. Exemplary packaging and producer cells are from 293, A549 or HeLa cells. AAV vectors are purified and formulated using standard techniques known in the art. Further suitable host cells (depending on the vector) are Chinese hamster ovary (CHO) cells, CHO dihydrofolate reductase deficient variants, such as CHO DX B11 or CHO DG44 cells (see, for example, Urlaub and Chasin. 1980. Proc. Natl. Acad. Sci. 77: 4216-4220), PerC.6 cells (Jones et al. 2003. Biotechnol. Prog. 19: 163-168) or Sp / 20 mouse melanoma cells (Coney et al. 1994. Cancer Res. 54: 2448-2455).

Retrovirus vector

Retroviral vectors can be used for gene delivery (Miller. 1992. Nature 357: 455-460). Retroviral vectors and more particularly lentivirus vectors can be used to practice the present invention. Thus, as used herein, the term "retrovirus" or "retroviral vector" is meant to include "lentiviral" and "lentiviral vector" respectively. Retroviral vectors have been tested and found to be suitable delivery vehicles for stably introducing the desired gene into the genome of a wide range of target cells. The ability of retroviral vectors to deliver unrearranged single copies of transgenes into cells makes retroviral vectors well suited for transgene transfer of genes into cells. Retroviruses are also introduced into host cells by binding of the retrovirus envelope glycoproteins to specific cell surface receptors on the host cells. As a result, the pseudotyped natural envelope protein is replaced by a heterologous envelope protein that has a different cell specificity than the native envelope protein (eg, binds to a different cell-surface receptor as compared to the native envelope protein). Retroviral vectors may also find utility in practicing the present invention. The ability to direct the delivery of retroviral vectors that encode one or more target protein coding sequences to specific target cells is desirable in practicing the present invention.

The present invention provides a retroviral vector comprising, for example, a retroviral delivery vector comprising one or more transgene sequences and a retroviral packaging vector comprising one or more packaging components. In particular, the present invention provides pseudotype retroviral vectors encoding heterologous or functionally modified envelope proteins for producing pseudotype retroviruses.

The core sequence of the retroviral vectors of the present invention can easily originate from a wide range of retroviruses including, for example, B, C, and D type retroviruses, and spumaviruses and lentiviruses. RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985). Examples of retroviruses suitable for use in the compositions and methods of the present invention include, but are not limited to, lentiviruses. Other retroviruses suitable for use in the compositions and methods of the present invention include Avian Leukosis Virus, Bovine Leukemia Virus, Murine Leukemia Virus, Mink-Cell Induced Virus ( Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis virus, and Raus sarcoma virus. Particularly preferred murine leukemia viruses are 4070A and 1504A (Hartley and Rowe. 1976. J. Virol. 19: 19-25), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245, Graffi, Gros (ATCC No. VR-590), Kirsteni Harvey Sarcoma Virus and Rauscher (ATCC No. VR-998. ), And Moloney Murine Leukemia Virus (ATCC No. VR-190). Such retroviruses are readily available from depository or collection agencies such as the American Type Culture Collection (ATCC, Manassas, VA) or can be isolated from known sources using commercially available techniques. Others are commercially available.

In one embodiment, the retroviral vector sequences of the invention may originate from lentiviruses. Preferred lentiviruses are human immunodeficiency viruses, such as type 1 or type 2 (ie HIV-1 or HIV-2, where HIV-1 is already associated with lymphadenopathy virus 3 (HTLV-III) and Called acquired immunodeficiency syndrome (AIDS) -associated virus (ARV)), or other virus associated with HIV-1 or HIV-2 identified as and related to AIDS or ADIS-like disease. Other lentiviruses include sheep Visna / maedi virus, feline immunodeficiency virus (FIV), bovine lentivirus, simian immunodeficiency virus (SIV), equine infectious leukemia virus (EIAV), and goat arthritis Encephalitis virus (CAEV).

Suitable genes and strains of retroviruses are well known in the art (see, eg, Fields Virology, Third Edition, edited by BN Fields et al. 1996. Lippincott-Raven Publishers, see: See, eg, Chapter 58, Retroviridae: The Viruses and Their Replication, Classification, pages 1768-1771, including Table 1 thereof. Retroviral packaging systems for producing producer cells and producer cell lines that produce retroviruses, and methods of making such packaging systems are also known in the art.

Typical packaging systems include at least two packaging vectors: a first packaging vector comprising a gag, pol, or a first nucleotide sequence comprising gag and pol genes; And a second packaging vector comprising a second nucleotide sequence comprising a heterologous or functionally modified envelope gene. Retroviral components may originate from lentiviruses such as HIV. The vector may lack functional tat genes and / or functional accessory genes (vif, vpr, vpu, vpx, nef). The system may further comprise a third packaging vector having a nucleotide sequence comprising a rev gene. The packaging system can be provided in the form of a packaging cell containing the first, second, and optionally third nucleotide sequences.

In aspects, there is applicability to various expression systems, in particular those using eukaryotic cells and advantageously mammalian cells. When natural proteins are glycosylated, preferred embodiments may include expression systems that will provide natural-like glycosylation for the expressed protein.

Lentiviruses generally include envelope glycoproteins SU (gp120) and TM (gp41) encoded by the env gene; CA (p24), MA (p17) and NC (p7-11) encoded by the gag gene; It shares several structural virion proteins, including RT, PR and IN, encoded by the pol gene. HIV-1 and HIV-2 contain auxiliary and other proteins involved in the regulation of the synthesis and processing of viral RNA and other replication functions. Auxiliary proteins encoded by vif, vpr, vpu / vpx, and nef genes can be excluded (or inactivated) from the recombinant system. In addition, tat and rev can be excluded or inactivated by, for example, mutations or deletions.

First generation lentiviral vector packaging systems provide separate packaging constructs for gag / pol and env, and typically use heterologous or functionally modified envelope proteins for safety reasons. In the second generation lentiviral vector system, accessory genes, vif, vpr, vpu and nef are deleted or inactivated. Third generation lentiviral vector systems are those from which the tat gene is deleted or otherwise inactivated (eg, via a mutation).

Compensation for transcriptional regulation generally provided by tat may be provided by the use of potent constitutive promoters such as human cytomegalovirus immediate early (HCAAV-IE) enhancer / promoter. Other promoters / enhancers are based on the strength of constitutive promoter activity, specificity to the target tissue (eg, liver-specific promoters), or other factors related to desirable control over expression, as understood in the art. Can be selected. For example, in some embodiments, it is desirable to use an inducible promoter such as tet to achieve controlled expression. Genes encoding rev are provided in separate expression constructs so that a representative third generation lentiviral vector system can include one and four delivery vectors for each of the four plasmids: gagpol, rev, envelope. Regardless of the generation of packaging system used, gag and pol may be provided on a single construct or on separate constructs.

Typically, the packaging vector is included in the packaging cell and introduced into the cell via transfection, transduction or infection. Methods for transfection, transduction or infection are well known to those skilled in the art. Retroviral delivery vectors of the invention can be introduced into a packaging cell line by transfection, transduction or infection to produce a producer cell or cell line. The packaging vector of the present invention can be introduced into human cells or cell lines by standard methods including, for example, calcium phosphate transfection, lipid infection or electroporation. In some embodiments, the packaging vector is introduced together using a predominant selectable marker, such as neo, dihydrofolate reductase (DHFR), glutamine synthetase or ADA, into the cell, followed by selection and in the presence of an appropriate drug. Entails isolation of the clones. The selectable marker gene may be physically linked to the gene encoded by the packaging vector.

Suitable cell lines are known which are structured such that the packaging function is expressed by suitable packaging cells. For example, US Pat. No. 5,686,279, which describes packaging cells; And Ory et al. 1996. Proc. Natl. Acad. Sci. 93: 11400-11406. Additional techniques for stable cell line production can be found in Dull et al. 1998. J. Virol. 72 (11): 8463-8471; And Zufferey et al. 1998. J. Virol. 72: 9873-9880.

Zufferey et al. 1997. Nat. Biotechnol. 15: 871-75 teach a lentiviral packaging plasmid wherein the 3 'sequence of pol comprising the HIV-1 envelope gene is deleted. The construct contains tat and rev sequences and the 3 'LTR is replaced with a poly A sequence. 5 'LTR and psi sequences are replaced with other promoters, such as inducible. For example, CMV promoters or derivatives thereof can be used.

Packaging vectors can contain additional changes to packaging function to enhance lentiviral protein expression and improve safety. For example, all of the HIV sequences on top of the gag can be removed. In addition, the lower sequence of the envelope can be removed. In addition, steps can be performed to modify the vector to improve splicing and translation of the RNA.

Optionally, a conditioned packaging system, such as described in Dull et al. 1998, supra, is used. In addition, self-improvement of the vector's biosafety by deletion of HIV-1 long terminal repeats (LTR) as described, for example, in Zufferey et al. 1998. J. Virol. 72: 9873-9880. The use of an inactivation vector (SIN) is preferred. Inducible vectors can also be used, for example, via tetracycline-induced LTR.

Promoter

In aspects, the vectors of the invention typically comprise heterologous control sequences, which include cytomegalovirus (CMV) imideate early promoter, RSV LTR, MOMLV LTR, and PGK promoters; heterologous control sequences, including but not limited to, constitutive promoters such as mTTR, TK, HBV, hAAT, tissue or cell type specific promoters including modulators or inducible promoters, enhancers, and the like.

Certain useful promoters include the LSP promoter (III et al. 1997. Blood Coagul. Fibrinolysis 8S2: 23-30), the EF1-alpha promoter (Kim et al. 1990. Gene 91 (2): 217-23) and Guo et al. 1996. Gene Ther. 3 (9): 802-10. Most preferred promoters are elongation factor 1-alpha (EF1a) promoter, phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus imidate early gene (CMV) promoter, chimeric liver-specific promoter (LSP), cytosine Megalovirus enhancer / chicken beta-actin (CAG) promoter, tetracycline reactive promoter (TRE), transthyretin promoter (TTR), simian virus 40 (SV40) promoter and CK6 promoter. An advantageous promoter useful in the practice of the present invention is the adenovirus major rate promoter (Berkner and Sharp. 1985. Nucl. Acids Res. 13: 841-857). Structural and functional information of related promoters is known in the art. Related sequences can be readily obtained from public databases and incorporated into vectors for use in practicing aspects of the present invention.

Particularly preferred promoters in the practice of the present invention are adenovirus major rate promoters. The expression cassette is a 5 'to 3' direction, a tripartite leader sequence, a self processing sequence, or a protease cleavage sequence that is operative for the adenovirus major rate promoter, the first coding sequence for the protein of interest or protein chain of interest. Followed by a sequence encoding a protein, a second coding sequence for the protein or protein chain of interest, and optionally a sequence encoding a self processing sequence or protease cleavage sequence, followed by a third coding sequence of the protein or protein chain of interest. Can be. All of these coding sequences are covalently linked and present in the same reading frame so that translation is not terminated in the polyprotein coding sequence. Self-processing or proteolytic processing during protein synthesis or after completion of the synthesis of the multipeptide cleaves the polyprotein into appropriate protein chains or proteins. In the case of immunoglobulin synthesis, the coding sequence for the light chain is present twice in the polyprotein coding sequence. Advantageously, the leader sequence coding region can be associated with a protein or protein chain sequence; Processing with signal peptidase may have the added advantage of removing certain residual amino acid residues at the N-terminus of the protein below the processing site. Components for immunoglobulin heavy chains include Met, protein initiated methionine; HC, heavy chain; LC, light chain, SPPC, self-processing or protease cleavage site. Expression constructs for immunoglobulin synthesis may include: Met-protease-SPPC-HC leader sequence-HC-SPPC-LC leader sequence-LC-SPPC-LC leader sequence-LC; Met-protease-SPPC-LC leader sequence-LC-SPPC-LC leader sequence-LC-SPPC-HC leader sequence-HC; Met-protease-SPPC-LC leader sequence-LC-SPPC-HC leader sequence-HC-SPPC-LC leader sequence-LC; HC leader sequence-HC-SPPC-LC leader sequence-LC-SPPC-LC leader sequence-LC; LC leader sequence-LC-SPPC-HC leader sequence-HC-SPPC-LC leader sequence-LC; LC leader sequence-LC-SPPC-LC leader sequence-LC-SPPC-HC leader sequence-HC; Met-protease-SPPC-HC leader-HC-SPPC-LC leader-LC.

Biotherapeutic Molecules Including Antibodies

Within the scope of the present invention, specifically expressed antibodies (immunoglobulins) are in particular processed antibodies corresponding to and / or originating tumor necrosis factor [HUMIRA / D2E7 (Abbott Biotechnology Ltd., Hamilton, Bermuda) Interleukin-12 (engineered antibody originating from ABT-874); interleukin-18 (engineered antibody originating from ABT-325); recombinant erythropoietin receptor (engineered antibody originating from ABT-007); Or E / L selectin (a processed antibody derived from EL246-GG) The coding and amino acid sequences of the processed polyproteins are described herein or may be used in the art. Antibodies include, for example, remicade (infliximab); rituxan / mabterra (rituximab); herceptin (trastuzumab); avastin (bevacizumab); cinagis (palivizumab); erbi Tux Shimab); Leopro (Abkicksimab); Orthoclonal OKT3 (Muromonab-CD3); Jenafax (Daclixumab); Seelect (Basiclikmab); Mylotarg (Gemtuzumab) ; Campathes (alemtuzumab); zevalin (ibritumomab); xsolaire (omalizumab); bexsar (tositumomab); and raftiva (epalizooma); Generally, the trademark-product name follows each generic name in parentheses: Further suitable proteins are, for example, one or more epoetin alpha, epoetin beta, etanercept, darbepoetin alpha, filgrastim, interferon beta 1a, interferon beta 1b, interferon alpha-2b, insulin glargine, somatropin, teriparatide, polytropin alpha, dornase, factor VIII, factor VII, factor IX, imiglucerase, necitrides, Renograstim and Von Willebrand factor; One or more generic names may each correspond to one or more trademark-product names of the product Other antibodies and proteins are also suitable for the present invention as is understood in the art.

The present invention also contemplates the controlled expression of coding sequences for two or more desired polypeptides or proteins or proproteins. Genetic control systems are useful for the controlled expression of specific genes or genes. In one exemplary attempt, a gene regulatory system or switch comprises a chimeric transcription factor having a ligand binding domain, a transcriptional activation domain and a DNA binding domain. The domain can be obtained from virtually any source and can combine any of a number of ways to obtain new proteins. The regulated gene system also includes a DNA response component that interacts with chimeric transcription factors. Such transcriptional regulatory components are located adjacent to the gene to be regulated.

Exemplary transcriptional control systems that can be used to practice the invention include, for example, the Drosophila ecdysone system (see Yao et al. 1996. Proc. Natl. Acad. Sci. 93: 3346). ), Bombyx ecdysone system (Suhr et al. 1998. Proc. Natl. Acad. Sci. 95: 7999), GeneSwitch using RU486 as an inducer (Texas) Trademark of Valentis, Woodland] Synthetic progesterone receptor system (Osterwalder et al. 2001. Proc. Natl. Acad. Sci. USA 98 (22): 12596-601); Tet and RevTet systems [tetracycline regulated expression systems] that use small molecules such as tetracycline (Tc) or analogs, for example doxycycline, to regulate (turn on or turn off) transcription of the target BD Biosciences Clontech, Mountain View, Calif., Knott et al. 2002. Biotechniques 32 (4): 796, 798, 800; Each of these includes ARIAD regulation techniques (Ariad, Cambridge, Mass.) Based on the use of small molecules that come with two intracellular molecules linked to transcriptional activators or DNA binding proteins. When these components come together, the transcription of the gene of interest is activated. Ariads have a system based on homodimers and a system based on heterodimers. Rivera et al. 1996. Nature Med. 2 (9): 1028-1032; Ye et al. 2000. Science 283: 88-91.

Aspects of an expression vector construct of the invention comprising a nucleic acid sequence encoding a antibody or fragment thereof or another heterologous protein or pro-protein in the form of a self-processing or protease-cleaved recombinant multipeptide may be used as an external therapeutic or transplant The gene can be introduced intracellularly, for example in vitro, ex vivo or in vivo for somatic cell delivery, or intracellularly in the production of recombinant polypeptides by vector-transduced cells.

Delivery of Host Cells and Vectors

Vector constructs of the invention can be introduced into suitable cells in vitro or ex vivo using standard methodologies known in the art. Such techniques include, for example, transfection with calcium phosphate, microinjection into cultured cells (see Capecchi. 1980. Cell 22: 479-488), electroporation (see Shigekawa et al. 1988. BioTechnology 6: 742-751), liposome-mediated gene delivery (Mannino et al. 1988. BioTechnology 6: 682-690), lipid-mediated transduction (Feigner et al. 1987. Proc. Natl. Acad. Sci USA 84: 7413-7417), and nucleic acid delivery using high-velocity microprojectiles (Klein et al. 1987. Nature 327: 70-73).

For in vitro or ex vivo expression, any cell effective for expressing the functional protein product can be used. Many examples of cells and cell lines used for protein expression are known in the art. For example, prokaryotic and insect cells can be used for expression. Eukaryotic microorganisms such as yeast can also be used. Expression of recombinant proteins in prokaryotic, insect and yeast systems is generally known in the art and can be adjusted for antibody or other protein expression using the compositions and methods of the present invention.

Examples of cells useful for expression also include mammalian cells, such as fibroblasts, cells from non-human mammals, such as cells from birds, pigs, murine and bovine cells, insect cells, and the like. . Specific examples of mammalian cells include COS cells, VERO cells, HeLa cells, Chinese hamster ovary (CHO) cells, CHO DX B11 cells, CHO DG44 cells, PerC.6 cells, Sp2 / 0 cells, 293 cells, NSO cells, 3T3 fibroblasts, W138 cells, BHK cells, HEPG2 cells, and MDCK cells, including but not limited to.

Host cells are cultured in conventional nutrient media suitably modified to induce a promoter, select a transformant, or amplify a gene encoding a sequence of interest. Mammalian host cells can be cultured in a variety of media. Commercially available media such as Ham's F10 (manufactured by Sigma), minimum mandatory medium (MEM) (manufactured by Sigma), RPMI 1640 (manufactured by Sigma), minimum mandatory medium (MEM) alpha medium, and two Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) is typically suitable for culturing host cells. Provided media generally will optionally include hormones and / or other growth factors (eg, insulin, transferrin or epidermal growth factor), salts (eg, sodium chloride, calcium, magnesium and phosphate), buffers (eg, HEPES), nucleosides (eg adenosine and thymidine), antibiotics, trace components, and glucose or equivalent energy sources. Any other necessary supplements may also be included at appropriate concentrations well known to those skilled in the art. Appropriate culture conditions for particular cell lines, such as temperature, pH, etc., are available, for example, on the Internet in the ATCC catalog (“atcc.org/SearchCatalogs/AIICollections.cfm” or as directed by a commercial supplier). ) Are generally known in the art, along with suggested culture conditions for the cultivation of multiple cell lines.

Expression vectors may be administered in vivo via various pathways (eg, intradermal, intravenous, intratumoral, intracranial, intraportal, intraperitoneal, intramuscular, in the bladder, etc.) to connect a number of self-processing cleavage sequences. By delivering a gene, two or more proteins or polypeptides can be expressed in an animal model or a human subject. Depending on the route of administration, therapeutic proteins exert their effects locally (in the brain or bladder) or systemically (other routes of administration). The use of tissue specific promoter 5 'for transcription translation frame (s) results in tissue specific expression of the protein or polypeptide encoded by the entire transcription translation frame.

Various methods of introducing recombinant expression vectors that carry transgenes into target cells in vitro, ex vivo or in vivo have already been described and are well known in the art. The present invention relates to therapeutic methods, vaccines and vaccines by infecting a targeted cell with a recombinant vector containing coding sequences for two or more desired proteins or polypeptides and expressing the protein or polypeptide in the targeted cells. Provide cancer therapy.

For example, in vivo delivery of recombinant vectors of the invention can be targeted to a wide variety of organ types, including but not limited to brain, liver, blood vessels, muscle, heart, lung and skin.

In the case of ex vivo gene delivery, target cells are removed from the host and genetically modified in the laboratory using recombinant vectors of the invention and methods known in the art.

Recombinant vectors of the invention can be administered using conventional manners, including but not limited to the forms described above. Recombinant vectors of the invention may be in various formulations including, but not limited to, liquid solutions and suspensions, microvesicles, liposomes, and injectable or injectable solutions. Preferred forms depend on the mode of administration and therapeutic application.

In aspects, the advantages of expression vector constructs in the production of immunoglobulins or other biologically active proteins in vivo include long-term administration of a single vector and sustained antibody expression in patients; In vivo expression of an antibody or fragment thereof (or other biologically active protein) with sufficient biological activity; And natural post-detoxification modifications of antibodies produced in human cells. Preferably, the expressed protein is identical or sufficiently identical to a naturally occurring protein such that the immunological response is not reduced or triggered, wherein the expressed protein is administered in many cases in a patient in need of said protein. Or continuously expressed.

Embodiments of the recombinant vector constructs of the present invention find additional utility in the in vitro production of recombinant antibodies and other biologically active proteins for use in therapy or research. Recombinant protein production methods are well known in the art and can be used for expression of recombinant antibodies using the self processing cleavage sites or other protease cleavage site-containing vector constructs described herein.

In one aspect, the invention provides a method for producing a recombinant immunoglobulin or fragment thereof by introducing an expression vector as described above into a cell to obtain a transfected cell, wherein the vector is in the 5 'to 3' direction. By: a promoter operably linked to the coding sequence for the immunoglobulin heavy and light chains or fragments thereof, the self processing sequence between each of the chains. The coding sequence for the immunoglobulin heavy chain or the coding sequence for the immunoglobulin light chain can be 5 '(ie, first) to the self processing sequence in the provided vector construct.

In an embodiment of a construct for an antibody, the sequence encoding the first or second chain for the antibody or immunoglobulin or fragment thereof comprises a heavy chain or fragment thereof derived from IgG, IgM, IgD, IgE or IgA. As broadly described, the sequences encoding chains for antibodies or immunoglobulins or fragments thereof also include light chains or fragments thereof from IgG, IgM, IgD, IgE or IgA. Aspects of the invention correspond to proteins for modified or derived forms thereof, including whole antibody molecules and other antigen recognition molecule fragments such as, for example, Fab, single-chain Fv (scFv) and F (ab ') ₂ It is about genes. Antibodies and fragments are animal-derived, human-mouse chimera, humanized, altered by Deimmunisation ™ (Biovation Ltd), altered to change affinity for Fc receptors, or complete It can be a person. Embodiments of ligand-binding molecules can mature affinity as understood in the art. In preferred embodiments, the antibody or other recombinant protein does not elicit or minimally elicit an immune response in the human or animal to which it is administered.

Antibodies can be bispecific and include, but are not limited to, diantibody, quadroma, mini-antibodies, ScBs antibodies, and knobs-into-holes antibodies.

Production and recovery of the antibody itself can be accomplished in a variety of ways well known in the art (Harlow et al. 1988. Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory). Other target proteins are collected and / or purified and / or used according to methods well known in the art.

In practicing embodiments of the present invention, the production of antibodies or variants thereof (analogues) using recombinant DNA technology can be achieved by culturing modified recombinant host cells under culture conditions appropriate for growth of the host cells and expression of coding sequences. Can be. To monitor the success of expression, standard techniques such as ELISA, RIA, and the like can be used to monitor the concentration of the antibody with respect to the antigen. Antibodies are recovered from the culture supernatant using standard techniques known in the art. Purified forms of these antibodies include, but are not limited to, affinity chromatography involving a Protein A, Protein G or Protein L column, or even specific epitopes of antigens associated with, or even requiring, specific antigens. It can be easily prepared by standard purification techniques. Antibodies can also be purified as understood in the art using conventional chromatography, such as ion exchange, hydrophobic interactions, affinity or size exclusion columns. See “Low pH hydrophobic interaction, which describes purification techniques. US Patent No. 5,641,870 to Rinderknecht et al. on June 24, 1997, entitled "chromatography for antibody purification," and September 2008, entitled "Process for purifying proteins in a hydrophobic interaction chromatography flow-through fraction." US Pat. No. 7,427,659 to Shukla et al. Purification techniques can be performed in combination with other techniques such as ammonium sulfate precipitation and size-limited membrane filtration or in various combinations. If the expression system is designed to include a signal peptide, the antibody obtained is secreted into the culture medium or supernatant; Intracellular production is also possible. Intracellular contents can be recovered and applied to the purification.

The desired level of processing of the polyprotein, for example cell culture conditions that facilitate the most complete processing, can be selected. Such processing occurs intracellularly, but can also occur extracellularly during or after the cell culture process.

Production and selection of antigen-specific, fully human monoclonal antibodies from mice processed with human Ig loci have already been described (Jakobovits et al. 1998. Advanced Drug Delivery Reviews 31: 33-42; Mendez et al. Nature Genetics 15: 146-156; Jakobovits et al. 1995. Curr Opin Biotechnol 6: 561-566; Green et al. 1994. Nature Genetics Vol. 7: 13-21).

High concentration expression of therapeutic monoclonal antibodies has been achieved in the milk of transgenic goats, and it has been found that antigen binding levels are equivalent to those of monoclonal antibodies produced using conventional cell culture techniques. The method is based on the development of a human therapeutic protein in the milk of a transgenic animal that carries genetic information to express the human therapeutic protein in its own milk. Once they are produced, these recombinant proteins can be effectively purified from milk using standard techniques. See, eg, Pollock et al. 1999. J. Immunol. Meth. 231: 147-157 and Young et al. 1998. Res Immunol. 149 (6): 609-610]. Animal milk, egg whites, blood, urine, seminal plasma and silkworm skewers from transgenic animals have proven potential as sources for the production of recombinant proteins on an industrial scale. Houdebine L M. 2002. Curr Opin Biotechnol 13: 625-629; Little et al. 2000. Immunol Today, 21 (8): 364-70; And Gura T. 2002. Nature, 417: 584-5860. The present invention is directed to a transgenic animal expression system for the expression of recombinant antibodies or variants (analogs) or other protein (s) of interest using the self-processing cleavage site-encoding and / or protease recognition site vectors of the present invention. Consider using it.

Successful production of recombinant proteins in plants including, but not limited to, potatoes, tomatoes, tobacco, rice, and other plants transformed by Agrobacterium infection, biolistic transformation, protoplast transformation, and the like Has been. Expression of recombinant human GM-CSF in seeds of transgenic tobacco plants, and expression of antibodies comprising Japanese chain antibodies in plants has been demonstrated. See, eg, Streaffield and Howard. 2003. Int. J. Parasitol. 33: 479-93; Schillberg et al. 2003. Cell Mol Life Sci. 60: 433A5; Pogue et al. 2002. Annu. Rev. Phytopathol. 40: 45-74; And McCormick et al. 2003. J Immunological Methods, 278: 95-104. The present invention contemplates the use of a transgenic plant expression system for the expression of recombinant immunoglobulins or fragments thereof or other protein (s) of interest using the protease cleavage site or self-processing cleavage site-encoding vector of the invention. .

In the context of insect cells, baculovirus vector expression systems have also been attempted as viable platforms for recombinant protein production. Baculovirus vector expression systems have been reported to provide advantages with respect to mammalian cell culture, such as ease of culture and higher expression levels (see, eg, Ghosh et al. 2002. Mol Ther. 6: 5- 11, and Ikonomou et al. 2003. Appl Microbiol Biotechnol. 62: 1-20). The present invention also contemplates the use of baculovirus vector expression systems for the expression of recombinant immunoglobulins or fragments thereof using the self-processing cleavage site-encoding vectors of the invention. Baculovirus vectors and suitable host cells are well known in the art and commercially available.

Yeast-based systems also express expression of recombinant immunoglobulins or fragments thereof or other protein (s) of interest, including two- or three-hybrid systems using the self-processing cleavage site-encoding vectors of the present invention. (See, eg, US Pat. No. 5,643,745, incorporated herein by reference).

Expression cassettes and vectors and recombinant host cells of the present invention comprising coding sequences for self-processing peptides alone or with additional coding sequences for proteolytic cleavage sites, many of which are known in the art and Examples of usefulness in the expression of protein components, biologically active proteins, proproteins and recombinant immunoglobulins or fragments thereof, of two- and three-hybrid systems in any protein expression system described herein are found. do. One skilled in the art can readily adapt aspects of the vectors, host cells, and methods of the invention for use in any protein expression system.

Example 1.Lon Protease Inteins and Expression Constructs

The three ATP-dependent Lon protease inteins are found in the New England Biolabs (NEB, Ipswich, Mass., USA) in the Intein Database and Registry; at http: // www. neb.com/neb/Inteins.html), Perler, FB (2002). InBase, the Intein Database. Nucleic Acids Res. 30, 383-384. These inteins originated from the organisms Pabi Lon Intein, Pyrococcus Prio Lon Intein and Pyrococcus horicosy OT3 (Pho Lon Intein). These Lon inteins have a proposed endonuclease domain, lysine instead of histidine as the second residue at the end of the intein, and different lengths (333, 401, and 474 amino acids, respectively). In the NEB database, all three lon inteins are shown as theoretical inteins, indicating that, according to the database, the presence of spliced products by the list contributors has not been demonstrated for the intein introduction provided. The endonuclease domain of the Pab Lon intein has been found to be experimentally inactive (see Saves I, Morlot C, Thion L, Rolland JL, Dietrich J, Masson JM. Investigating the endonuclease activity of four Pyrococcus abyssi Inteins. Nucleic Acids Res. 2002 Oct 1; 30 (19): 4158-65.

We have found that the inteins contained in the ATP-dependent protease lon family of genes are very effective in mediating cleavage of antibody heavy and light chains in the design of a variety of single transcription translation frame constructs. Sequence information related to these inteins is provided herein.

Lon intein sequence and vector construct design

Table 1 provides protein sequence information for the Pab Lon intein, Accession No. CAB50486.1 from the NCBI / protein, PAB1313 Pab Lon intein, including the −1 and +1 extein residues (SEQ ID NO: 1) .

Table 2 describes the nucleotide sequences for the Pab Lon intein optimized for codon use.

Table 3 describes the protein sequence encoded by SEQ ID NO: 2, SEQ ID NO: 3.

Table 4 provides protein sequence information for the NCBI / protein, Pfu Lon intein in PF0467, Accession No. AAL80591.1, including the −1 and +1 extein residues (SEQ ID NO: 4).

Table 5 provides nucleotide sequence information for the native Pfu Lon intein (SEQ ID NO: 5).

Table 6 describes the protein sequence encoded by SEQ ID NO: 5, SEQ ID NO: 6.

Pfu Lon inteins were cloned using PCR techniques. Pab Lon intetan nucleotide sequences were designed and synthesized according to mammalian codon usage. The protein sequence of the Pyrococcus abisii lon protease intein is publicly entrusted intein sponsored by New England Biolabs, Ipswich, Massachusetts (http://www.neb.com/neb/Inteins.html) Obtained from Inbase, which is a database of. The protein sequence obtained is listed as EMBL Accession No. CAB50486.1, gi5459000; The protein sequences listed on the website were used. Pab-lon intein protein sequences are shown in Table 7.

The Pab-lon protein sequence was back-decoded using a method registered by GeneArt (GeneArt AG, Regensburg, Germany) with a DNA sequence optimized for mammalian expression. The Pab-lon intein DNA sequences obtained are shown in Table 8. DNA constructs may optionally provide additional flanking modulators depending on the selection of specific cloning and expression vectors and the corresponding molecular biology attempts using conventional techniques. DNA sequences were synthesized as 999 bp fragments (GeneArt Synthesis Number 0611467) and transferred into the GeneArt vector plasmid, pGA4 (see: The Registry of Biological Standard Parts at http://partsregistry.org, including Part: BBA_J70003). The provided DNA material was resequenced and determined to correspond to the designed sequence. This DNA material was used directly as a source / template for subsequent Pab-lon intein containing plasmid constructs; The plasmid did not reproliferate.

The following mammalian expression vectors were constructed: pTT3-pfu lon HL (+), pTT3-pfu lon HL (-), pTT3-pfu lon LH (+), pTT3-pfu lon LH (-), pTT3-pfu lon LKH (+), pTT3-pfu lon LKH (-), pTT3-pab lon HL (+), pTT3 pab lon HL (-), pTT3-pab lon LH (+), pTT3-pab lon LH (-), pTT3- pab lon LKH (+), pTT3-pab lon LKH (-). Here, the H and L components represent immunoglobulin heavy and light chains for antibodies designed with D2E7. For a schematic representation of the pTT3 pablon HL (−) construct, reference is made to FIG. 1. FIG. 2 shows aspects of the structures for the sORF components of these transient expression vectors capable of expressing D2E7 antibodies.

Although the pTT3 vector represents a particular embodiment, additional embodiments may include aspects of an isolated nucleic acid encoding one or more proteins described herein. A further aspect provides a vector comprising an isolated nucleic acid sequence, wherein the vector is pcDNA; pTT (Durocher et al., Nucleic Acids Research 2002, Vol 30, No. 2: E9); pTT3; pEFBOS (Mizushima, S. and Nagata, S., 1990, Nucleic Acids Research Vol 18, No. 17: 5322); pBV; pJV; And pBJ. As noted above, various constructs were made in the pTT3 vector backbone. The vector has an EBV replication origin, which allows for its episomal amplification in 293E cells (which express Epstein-Barr virus nuclear antigen 1) transfected in suspension culture (see Durocher describing vector pTT). et al.). With respect to pTT, pTT3 has an additional multiple cloning site as shown in US Patent Application Publication No. 20050147610 by Ghayur, Tariq et al. on July 7, 2005.

Each pTT3-based vector had one ORF regulated by the CMV promoter. In ORF, the intein sequence was inserted in the frame between the antibody heavy and light chains (HC and LC, or simply H and L, respectively) in the order of HC-intein-LC or LC-intein-HC. Constructs with the name "HL" have an antibody HC coding sequence followed by an intein followed by an LC coding sequence; Constructs with the name “LH” have an LC coding sequence followed by an intein and then an HC coding sequence. Constructs with the name “LKH” have lysine (K) inserted between the LC and the intein. The construct with the name “(−)” has methionine inserted between one signal peptide and the last amino acid of the intein and the first amino acid of the mature antibody heavy or light chain following the intein at the beginning of the ORF. Constructs with the name “(+)” have one signal peptide at the beginning of the ORF and a second signal peptide at the beginning of the antibody subunits below the intein.

The construct was introduced into 293E cells via transient transfection. In summary, the complex was prepared using an ORF construct and a pTT3 vector encoding polyethyleneimine (PEI). PEI-DNA complexes were used to transfect HEK293E cells (Durocher et al., 2002, Nucl. Acids Res. 30: E9). Cells and culture supernatants were collected 4-7 days after transfection for analysis.

Protein Expression by Constructs. In many transient expression experiments, culture supernatant samples were collected 7 or 8 days after transfection. Samples were evaluated by ELISA and contained concentrations or ranges of antibodies secreted from the measurement of IgG as shown below.

A conventional two-vector system expressing the same antibody as the series of constructs described above and using the same regulatory components was included in the experiment as a control (Table, bottom column). Thus, the control vector expressed the D2E7 antibody using conventional attempts to introduce antibody heavy and light chains from two separate ORFs carried in two separate pTT3 vectors. The antibody secretion concentrations produced from this control vector system ranged from 10 to 60 μg / ml as shown in the table.

IgG secretion concentrations produced by several sORF construct designs using Lon intein are in the same range, or more, compared to those produced using conventional control vectors. This concentration is significantly higher than those produced using the "2A" technique reported to be 1.6 μg / ml in mammalian cells (Fang et al., 2005, Nature Biotechnology 23: 584-590). . Although both Pab Lon and Pfu Lon inteins were used in construct design to obtain the desired concentration of antibody production, the Pab Lon intein in the described pTT3 constructs allowed for higher concentrations of antibody secretion. These data also suggest that antibody secretion concentrations are generally higher when the HL construct design is used than when the LH construct design is used. By combining features of the sequence of the immunoglobulin chains and aspects of the signal sequence, the HL (−) construct was able to produce the maximum concentration of secreted antibody product among those studied.

Further Characterization of Expression Products

Certain sORF constructs listed in Table 9 were further characterized in terms of expression phases, including analysis of expression products. These constructs included four examples that produced relatively high levels of secreted antibodies: pTT3 pfu lon HL (-), pTT3 pfu lon HL (+), pTT3 pfu lon LKH (-), and pTT3 pab lon HL (-). Secreted antibodies produced from these constructs were purified by Protein A affinity chromatography and analyzed on both reducing and non-reducing SDS-PAGE gels and the N-terminal amino acid sequences for their HL and LC were determined.

Samples produced using pTT3 pfu lon HL (-), antibody HC, antibody LC, and fully, with distinguishable migration from antibodies produced by conventional methods using conventional vectors such as the control D2E7 vector described above Gel transfer bands corresponding to the assembled antibody (on the non-reducing gel) were contained. On the reducing gel, in addition to the bands corresponding to the antibodies HC and LC, two high molecular weights (MW) which appeared to correspond to unprocessed 3-part protein (HC-intein-LC) and partially processed HC-intein fusions There is also a band. This evaluation was based on western blot analysis and mass spectrometry analysis. The abundance of these two bands can be reduced by modifying the culture conditions and was believed to be dependent on the culture conditions. These high MW products can be conveniently removed from the fully processed antibody drug substance using methods according to other descriptions provided herein and / or as understood in the art.

Samples produced using pTT3 pfu lon HL (+) bands corresponding to antibody HC, antibody LC, and complete antibody (on non-reducing gel), with indistinguishable migration from antibodies produced by conventional methods It contained. In addition, there was one larger MW band corresponding to the 3-part polyprotein. Samples produced using pTT3 pfu lon LKH (-) also correspond to HC, LC, and complete antibodies (on non-reducing gels) with indistinguishable migration from antibodies produced using conventional vectors. It contained. On the reducing gel, in addition to the bands corresponding to HC and LC, there were also two higher MW bands. The first of these corresponded to a three part multiple protein, as described above for other vector designs; The second band corresponded to the LC-intein fusion product and resulted from incomplete cleavage at this junction. In terms of the relative abundance of the product, it was believed that many LC-intein fusions exist, such as truncated LCs.

Samples produced using pTT3 pablon HL (-) contained bands corresponding to HC, LC, and complete antibodies (on non-reducing gels) with indistinguishable migration from antibodies produced by conventional methods. . On the reducing gel, in addition to the bands corresponding to HC and LC, there was one major higher MW band that was considered to correspond to the unprocessed 3-part protein based on Western blot analysis. In comparison to samples produced using pTT3 pfu lon HL (−), there was a relatively smaller amount of this 3-part higher MW band. The results indicate that even if the Pfu lon intein and Pab lon intein are homogeneous and functionally similar in our vector design, Pab lon mediated N-terminal cleavage was observed after HC- expression observed from the Pab lon construct. Since little intein fusion is present, it is suggested that it is more complete than that mediated by Pfu lon. However, it is noted that both constructs can yield fully assembled antibody products.

On the other hand, certain protein yields, such as unprocessed and partially processed proteins, can be considered to be contaminated products compared to other construct yields, such as fully processed and fully self-assembled antibody products. On the other hand, this particular protein yield may be useful, for example, for further processing reactions and / or directed assembly materials that are not yet able to produce a complete antibody product. If these protein productions are observed as contaminated by-products, there are choices and attempts to facilitate removal, as noted, to concentrate or purify the desired component, such as a complete antibody.

In addition to extracellular samples of culture supernatants from various construct expression systems, intracellular samples were also obtained and analyzed by Western blot analysis using detection antibodies with specificity for both HC and LC. Similar protein species were observed as described for these species in cultured supernatant samples.

The N-terminal amino acid sequences of both the heavy and light chains of the various constructs were measured (see Table below). The results indicated that intein-mediated protein cleavage occurred precisely at two splicing connections, namely the linkage of HC and intein components and the linkage of LC and intein components.

Lon Intaine From the construct IgG1  Functional Properties of Antibodies

The D2E7 antibody product secreted from the sORF construct design of Table 9 was also analyzed by antigen-specific ELISA. The analysis demonstrated that the construct antibody product binds to human TNFalpha, the ligand of the D2E7 antibody. Thus, intein-based constructs and expression systems are capable of expressing and producing sORF products that yield functional and antigen-specific, fully self-assembled multimeric antibodies.

Antibodies produced using the pTT3 pfu lon HL (−) construct were purified by Protein A affinity purification followed by SEC, size exclusion chromatography. Purified antibodies were analyzed by surface plasmon resonance technology using the BiaCore ™ system. Characterization of the amount of sORF construct production involved its binding aspect to the related ligand, TNFα. In Table 11, the result of the values is the binding rate constant (ka, units of 1 / Ms); From BiaCore analysis on the mechanical parameters of dissociation rate constants (kd, units of 1 / s), and equilibrium dissociation constants (units of KD, M). Dissociation constant values (KD) are understood to be similar to those of adalimumumab (D2E7) antibodies produced using a convenient vector (with two distinct immunoglobulin chain sORFs).

Example  2. Light chain  To produce a modified antibody of the sequence sORF Construct

Several sORF constructs were generated with light chain sequences that are variants from the immunoglobulin light chain of D2E7. These sORF constructs can also be processed into heavy chains as in D2E7 to produce IgG1 antibody material. Using the constructs pTT3 pfu lon HL (-) and pTT3 pab lon HL (-) as the backbone, the inventors used a sequence variation at the C-terminal splicing linkage, ie, the linkage between the intein and the lower immunoglobulin light chain component. Constructs with were generated and tested (Table 12). Secreted immunoglobulins of certain constructs were purified by Protein A affinity purification and analyzed on reducing and non-reducing SDS-PAGE gels. Intracellular samples were also analyzed by Western blot analysis using antibodies against both HC and LC (see, eg, FIGS. 3 and 4).

3 shows the results of SDS-PAGE gels for protein analysis of sORF expression products. Secreted IgG molecules were purified by Protein A affinity chromatography and separated under non-reducing (A) and reducing conditions (B) on SDS-PAGE gels. Lanes and samples from left to right are as follows: (lane 1) MW marker; (2) control construct products, D2E7 antibodies from non-sORF expression systems; (3) Pab-lon mut A1; (4) Pab-lon mut A2; (5) pTT3 pfu lon YP, and (6) pTT3 pfu lon MA.

4 shows the results of SDS-PAGE gels for protein analysis of additional sORF expression products. Secreted IgG was purified by Protein A affinity chromatography and separated under non-reducing (A) and reducing (B) conditions on an SDS-PAGE gel. Lanes and samples from left to right are as follows: (lane 1) MW marker; (2) control D2E7 product; (3) pTT3 pfu lon HL (−); And (4) pTT3 pfu lon MutA.

Immunoglobulin secretion concentrations produced by the constructs of Table 12 are shown in Table 13. Amino acids are measured at the N-terminus of the mature light chain and the results of the characterized partial sequences are shown.

result

For these constructs, the use of different AA residues at the +1 position (right after intein) was considered a factor in the yield of secreted antibodies. The use of amino acid residues H, Y, R, V, Q, A, N, and M at this position yielded relatively higher levels of antibody expression. Analysis of the light chains for their N-terminal amino acids (Table 13) suggested complete and precise cleavage at the C-terminus of intein. Similar to the antibodies produced by the constructs pTT3 pfu lon HL (−) and pTT3 pab lon HL (−), processed HCs and LCs also assembled a complete antibody showing most of the secreted protein species. However, when amino acid aspartate (Asp; D) was used directly after intein as in construct pTT3 pfu lon MutB, there was little antibody secretion. Intracellular proteins produced by this construct were analyzed. When D is the first amino acid following intein, little cleavage occurs at the C-terminal splicing junction and a small amount of antibody LC and a relatively large amount of intein-LC fusion protein are obtained.

The amino acid sequence of the mature germ line light chain of the kappa isotype variable region (V _kappa ) generally begins with D, E, N, A, or V; The same type of the lambda (V _lambda) is generally initiated by the Q, S, L, or N. From the results of the inventors, embodiments of the sORF vector using Pab lon or Pfu lon intein for antibody production include those with LCs starting with any amino acid. In a preferred embodiment, for the purpose of achieving higher efficiency of overall complete processing, the LC starts with amino acids other than D or E, although such amino acids may be provided as an operational choice.

We have found that the region between intein and the underlying mature antibody LC from intein appears to contribute to cleavage efficiency at the N-terminal splicing junction. For example, we compared the results of the constructs pTT3 pfu lon HL (-) and pTT3 pfu lon MutA. When pTT3 pfu lon HL (-) is used, cleavage at the N-terminal splicing junction is not complete, but a partially partially processed HC-intein fusion protein is obtained and the amount of the protein species is construct pTT3 pfu lon Significantly decreases if MutA was used instead (see FIG. 2). Thus, while various constructs may be useful for producing the desired product, the particular construct may be capable of producing relatively higher yields of particularly desirable products, eg, fully processed and self-assembled multimeric secreted antibodies. Can contribute together.

Example  3. sORF Constructive Intaine  Additional Choices for Ingredients

Metanococcus Jannaskyi  And Pyrococcus From his father klbA  Gene Intaine

We further tested intein selection for sORF constructs that include the inteins of the klbA gene, such as from Metanococcus and Pyrococcus species. We have found that the inteins contained in the klbA gene are also efficient choices to mediate protein expression and processing, including in the context of cleavage of antibody heavy and light chains in the design of various single transcription translation frame constructs.

In particular, we tested klbA intein from Metanococcus jannaskyi (Mja klbA intein), Pyrococcus acciae (Pab klbA intein), and Pyrococcus puriosus (Pfu klbA intein). . The intein from the first two organisms is a mini-intein lacking an endonucleade domain, while Pfu klbA is a full sized intein. The sequence length of the native intein protein segment is 168, 333, and 522 amino acids, respectively. Sequence information regarding these inteins is provided in the table below. Thus, intaines, modified intaines, and constructs are developed for expression systems.

KlbA Intein Sequence and Vector Construct Design

The nucleotide sequence of Mja klbA was modified to allow for relative optimization of mammalian codon usage. Table 14 provides nucleic acid sequence information for the Mja klbA intein gene of Metanococcus jannaskyi thus modified (SEQ ID NO: 34). Table 15 provides protein sequence information for the Mja KlbA intein segment (see also NCBI / protein, Accession No. Q58191 in MJ0781). Table 16 provides nucleic acid sequence information for modified Pab klbA intein genes in terms of codon usage related to native sequences. For native sequences, see also accession number (NCBI / protein, B75050 in PAB1457) of NEB Inbase information for Pab KlbA intein. According to this source, the indicated protein amino acid sequences are shown to contain -1 and +1 extein residues which appear to be G and C, respectively. Table 17 provides amino acid sequence information for Pab KlbA intein protein segments. Table 18 provides nucleic acid sequence information for the Pfu klbA intein gene (natural). Table 19 provides amino acid sequence information for Pfu KlbA intein protein segments (see also Accession No. AE010211 from NCBI).

We synthesized the nucleotide sequences of the Mja klbA intein and Pab klbA intein using sequences using mammalian codon applications. The following mammalian expression vectors were constructed: pTT3- Pab klbA HL (−); pTT3- Pab klbA HL (+); pTT3- Pab klbA LH (−); pTT3- Mja klbA HL (−); pTT3- Mja klbA HL (+); pTT3- Mja klbA LH (−). These constructs were prepared in the PTT3 vector backbone, as also described herein. The Pfu klbA intein nucleotide sequence is a native sequence and pTT3-Pfu-klbA-HL (+) was also constructed.

As shown for constructs using the Lon protease intein, all constructs with the name "HL" have an antibody immunoglobulin heavy chain (HC) coding sequence followed by an intein segment and then a light chain (LC) coding sequence. Similarly, all constructs with the name "LH" have an antibody LC coding sequence followed by an intein segment and then an HC coding sequence. Constructs using the "(-)" designation are defined at the beginning of the ORF between the last amino acid of one signal peptide and intein segment and the first amino acid of the lower antibody segment, e.g., the mature antibody heavy or light chain following intein. Has methionine inserted in it. The construct with the name “(+)” has one signal peptide at the beginning of the ORF and a second signal peptide at the beginning of the antibody subunit below the intein.

Constructs with various KlbA intein segments and structures were introduced into 293E cells via transient transfection techniques. Seven to eight days after transfection, culture supernatants were analyzed by measuring IgG concentration using ELISA on secreted antibodies. See Table 20 for these results with values of microbial units per ml of sample for each construct expression system.

We have purified and analyzed secreted antibody products expressed by the constructs, pTT3-Pab klbA HL (−) and pTT3-Mja klbA HL (−). Antibody products were purified by Protein A affinity chromatography and characterized under conditions of both reducing and non-reducing by electrophoretic techniques of SDS-PAGE (see FIG. 5). Under non-reducing conditions, the culture supernatant samples from these two vectors migrated primarily as a single band but as a single band with apparently higher molecular weight compared to the control antibody. Under reducing conditions, we found that culture supernatant samples from these two vectors contained corresponding detectable bands in size for the antibody LC and HC-intein fusion components. The corresponding immunoblot with either antibody against IgG1 Fc or kappa light chain is consistent with the characterization of these bands. These results indicate that there is a relatively efficient cleavage at the C-terminal splicing connection, but the cleavage at the N-terminal splicing connection is not efficient or even nearly total. However, even for constructs and expression systems where less than complete cleavage efficiency is achieved, immunoglobulin heavy and light chain subunits could be assembled and secreted as complete IgG antibody molecules.

Modification of Klb Intein at N-terminal Intein Splicing Connection

In view of the results described above for cleavage at the N-terminal splicing connection, the inventors have made additional efforts to provide improved cleavage efficacy. The inventors noted that the first amino acid of each of these two inteins, Pab klbA and Mja klbA, is alanine (Ala; A) instead of cysteine (Cys; C). We understand that cysteine is a moiety that can function as a nucleophile in other intein systems. The inventors have introduced the effect of reintroduction of the nucleophilic amino acid, cysteine, at this position, at the end of the immunoglobulin HC segment, with one amino acid, glycine, which is natural to the klbA extein top of the intein, Tested. See Table 21, which provides sequence information for the protein segments for these additional constructs. The table shows three constructs with mutations at the native Pab klba intein, Pab klba HL (-) constructs (which are referred to in the context as WT), and at the N-terminal splicing junction: Pab klba HL ( -) GC; Pab klba HL (-) GA; And two splicing linkages to Pab klba HL (-) KC. Asterisk ( ^* ) indicates the position at which the variant amino acid residue was introduced into the mutant construct. Among these constructs, Pab-klbA HL (-) GC demonstrated the ability to express and process proteins with efficient cleavage at the N-terminal intein junction. See also FIG. 6, which depicts the expression of IgG proteins and the results of SDS-PAGE analysis from certain constructs.

Materials and Methods for the Generation of Vectors: Pab - klbA HL (-) Mutant GA , GC , And KC Construction of

Several variant forms of specific vector constructs were generated. Pab-klbA HL (-) mutants GA, GC, and KC were constructed by PCR. PCR product # 1 was generated by PCR amplification at the 3 'end of the heavy chain using forward primer HC-F and reverse primer Hint-R. Forward primers GA-F, GC-F, and KC-F contained complementary sequences at the 3 'end of the preferred mutations and heavy chains. PCR product # 2 was produced by amplification at the 5 'end of the Pab-klbA intein using primers GA-F, GC-F, or KC-F and reverse primer intein-R-2. Thereafter, PCR products # 1 and # 2 were purified using Qiagen Gel Extraction kit. Purified PCR products were annealed together and amplified using external primers HC-F and intein-R-2 to generate PCR product # 3. Vector Pab-klbA HL (-) was digested using restriction enzymes Sacll and Rssll and then purified using a Kiagen gel extraction kit. PCR product # 3 was subcloned by homologous recombination in maximal efficiency DH5α cells (Invitrogen) into Pab-klbA-HL (-) (cut into Sacll and Rssll). Transformants carrying the correct mutations were determined using colony PCR followed by sequence analysis. DNA from the correct clone was amplified and purified using the Qiagen Maxi kit. Primer sequences are shown in the table below.

Example  4. sORF Construct  Generation of expressing stable vectors and cell lines

Stable expression vectors and cell lines expressing such vectors can be developed using aspects of the sORF construct. As an example, stable expression vectors containing sORF with Pab Lon intein were stably transfected into CHO (Chinese Hamster Ovary) cell lines. We used the same ORF as in CMV enhancer, adenovirus major rate promoter, SV40 polyA sequence, gastrin transcription terminator, DHFR coding sequence driven by SV40 promoter, and pTT3 pablon HL (-) used in transient expression system. Stable sORF expression vectors with components were designed and constructed. Thus, the sORF construct pA190-Pab-lon HL (-) can be prepared and used as a stable expression vector; See FIG. Additional constructs are similarly prepared.

Using calcium phosphate transfection technology, the pA190 construct was introduced into CHO cells (designated CHO B3.2) and the cells were loaded with MEM and 5% FBS at a density of 200 cells / well in 48 96-well plates. Plated in the selection medium containing. Transfection plates were monitored for growth of cells / colonies and IgG secretion.

Thirty clones from the sORF stable expression vector pA190-Pab-lon HL (−) and 32 clones from the control stable expression vector pA190 transfection reaction were selected and grown. In this step, IgG secretion concentrations were evaluated for selected clones without amplification, and concentrations were also assessed after amplification using 20 nM methotrexate (MTX). For a stable expression system, the sORF vector is significant in growth positive wells (2304 out of 4608 total wells) with the appropriate number (443 out of 2304) and ratio, ie, 19% of the samples positive for IgG secretion. Frequency was generated. IgG secretion concentrations under conditions of 0 nM MTX in 12-well plates ranged from about 0.3 to about 2.5 μg per ml of culture supernatant for ˜29 selected clones. Under conditions using 20 nM MTX, IgG secretion concentrations for ˜24 selected clones ranged from about 0.1 to about 6 μg per ml using about one half of the cloned clones demonstrating a secretion concentration higher than 2 μg / ml. It was. It was noted that sORF construct clones demonstrated relatively rapid conformity in adherent culture vessels. For example, in adherent cultures with 20 nM MTX, SORF clones grew more rapidly and reached conformity faster than conventional vector clones. On one passage in 20 nM MTX, 28% of clones from conventional vectors reached consensus within 6 days (4, 5 or 6 days); Here, 77% of the clones from the sORF pab lon vector reached consensus within 6 days (see Figure 8). These data suggest a strong advantage of using sORF expression vectors as compared to conventional vectors in the development of stable expression systems involving CHO cell line development. The sORF clone also demonstrated favorable levels of antibody secretion under direct amplification conditions with 100 nM MTX. In the experiment, 16 clones exposed to 100 nM MTX obtained an average IgG secretion concentration of 6 μg / ml, the top five clones averaged 12 μg / ml and the production concentration of the top clone was 24 μg / ml. The table below shows the results of using MTX amplification of the maximum expression level in each amplification step. Values are μg per ml of IgG for various clones with pA190-Pab-lon-HL (−) constructs.

Materials and Methods for Stable Expression Systems

Chinese hamster ovary cells cultured in alpha MEM supplemented with H / T and 10% dialysis FBS were transfected with expression vectors using a calcium phosphate co-precipitation procedure. Kingston, R.E., et al. (1993), Unit 16.23: Amplification Using CHO Cell Expression vectors, Current Protocols in Molecular Biology (Ausubel, FM, Brent, R., Moore, DM, Kingston, RE, Seidman, JG, Smith, JA, and Struhl, K. , eds; Wiley Interscience, New York), 2: 16.23.1]. The following day, cells were transfected with trypsin / EDTA at room temperature and 5% dialyzed FBS (α-MEM + 5% dFBS), alpha supplemented with growth medium selective for transfected cells expressing DHFR from expression vector Resuspend in MEM. Culture supernatants surviving the selection were screened using ELISA specific for human IgG gamma chain. Cell lines providing the maximal ELISA signal were incubated in MTX containing α-MEM + 5% dFBS. MTX is an inhibitor of DHFR chosen for cells that produce higher concentrations of enzyme due to amplification of the vector. Cell lines were incubated in various concentrations of MTX and monitored for expression of antibodies.

In aspects, the compositions and methods of the present invention may employ pA205 vector constructs, or derivatives thereof, as described, for example, in US 20080241883, filed Oct. 2, 2008, to Gion et al. Can be.

Thus, stable cell expression systems are generated using various sORF designs and constructs. In particular, the sORF system is well suited for integration using the CHO platform for the expression of biological therapeutics such as antibody molecules.

Example 5 Characterization of Aspects to Intein C-Terminal Splicing Connections in sORF Constructs

We tested aspects of intein C-terminal splicing linkages in the context of sORF constructs. We have created about 40 new constructs in part to characterize aspects related to splicing linkage lengths that can affect cleavage efficiency at the first amino acid and C-terminal splicing linkages at the bottom of the intein. These additional constructs had mutations in the light chain linkage mutations. In general, we focused on residues at or near the N-terminal end of the light chain; Each of methionine (Met1) at position 1 and aspartate (Asp2) at position 2 was replaced with all 20 possible natural amino acid residues (see FIG. 9). These two series of light chain linkage mutant constructs used Pab Lon intein segments in the D2E7 antibody coding sequence and HC-intein-LC structure.

The construct was transfected into 293 cells. Transient expression resulted in high IgG titers using most of the Met substitution constructs, many of which yielded higher expression levels than control constructs having Met at this position (FIG. 10). Asp substitution constructs generally yielded lower levels of antibody expression (see FIG. 11).

The efficiency of polyprotein processing was tested (see, eg, FIG. 12). A library of constructs based on the variation of Met produced efficient processing of both HC and LC from polyproteins, similar to the Pab Lon HL (−) constructs described above. Libraries of constructs based on variations of Asp appear to have relatively impaired C-terminal processing and produced very small amounts of LC and significant amounts of intein-LC fusion protein species. This result of incomplete cleavage appears to be related to the total length difference of one amino acid unit as it is interpreted as being independent of amino acid properties at the splicing junction. However, it is noted that relatively inefficient constructs can still produce some IgG products.

In the experiment, IgG antibody products from 10 of 20 constructs were further analyzed in the methionine-variable library. Samples were batches purified using Protein A affinity chromatography and the light chain components were analyzed by mass spectroscopy, including evaluation of molecular weight. The results of mass spectrometry show specific constructs (Pab lon M1 A, Pab lon M1 D, Pab lon M1 E, Pab lon M1 F, Pab lon M1 G, Pab lon M1 H, Pab lon M1 I, Pab lon M1 K, It was shown that antibody light chains from Pab lon M1 L, and Pab lon M1 C) were formed as processed in construct design with precise cleavage occurring through intein-mediated reactions. Constructs where Cys was used and replaced with Met produced almost unprocessed HC and LC components and contained one protein species, which may be a splicing product, with some degree of Cys at the +1 position of the antibody light chain. It is proposed to support protein splicing. In all the antibody light chains produced, the presence of the first amino acid of the LC indicates that the antibody product produced using these vectors will be homogenous in the LC N-terminal region and processed by amino peptidase activity where the LC may be endogenous. Demonstrate sensitivity

Example  6. sORF Construct  Used antibodies ABT Expression of -847

We have developed sORF constructs adapted to express antibodies comprising human lambda light chains. We worked with ABT-847, a fully human antibody with specificity for the antigen, interleukin-12. The antibody has heavy and lambda isomeric light chains of human IgG1 isomorphism (see, eg, US Patent No. 6,914,128).

Five sORF constructs capable of expressing ABT-874 were prepared using homologous recombination. 13 shows the structure of SORF components of these constructs. Three of the constructs had the structure of HC-intein-LC, and two constructs had the structure of LC-intein-HC. These vectors were introduced into HEK293 cells via transient transfection and their antibody expression levels were assessed by IgG ELISA. The three HC-intein-LC constructs obtained titers of antibody in samples of culture supernatants with similar structure (HC-intein-LC) but similar to the constructs with D2E7 antibody fragments. In both of these cases, the ABT-874 and D2E7 sORF expression systems used the Pab Lon HL (−) phase. Two constructs with ABT-874 antibody segments in the LC-intein-HC structure yielded lower levels of IgG titers.

Samples of IgG produced in supernatants were batches purified by Protein A affinity chromatography and analyzed by SDS-PAGE electrophoresis. These analytes showed that the LC component was fully processed from the polyprotein in all three HC-intein-LC constructs.

IgG samples were also characterized using mass spectroscopy. This analysis confirmed that the LC produced from the three HC-intein-LC constructs started with the appropriate amino acid according to the construct design. The results are consistent with the precise cleavage mediated by the intaines used in the structure. The LC component produced from two constructs without additional methionine residues was identical to the material from the control sample of the ABT-874 antibody. Thus, although modifications can be achieved as performed in the case of D2E7 antibodies, such modifications are optional in terms of achieving the desired N-terminal LC amino acid sequence in the expression product from the described constructs. Mass spectrometry analysis was also used to evaluate the molecular weight of HC, demonstrating the MW predicted according to the construct design.

Example  7. Intaine -Based tablet tag sORF Construct

In aspects of the present invention, constructs are designed using inserts. In aspects of a construct with an insert, the insert can provide a detectable signal or is useful for providing a binding or recognition component. In aspects of the invention, constructs are designed to facilitate the separation of certain construct-related expression products from one or more of these products. For example, a vector design can be generated to allow purification of a fully processed, assembled multimeric antibody product from a mixture of components including a protein partially processed from an intein splicing reaction. With respect to the expression of the HL construct, the structure of H-intein-L can produce H, intein and L components by causing an incomplete cleavage reaction at either or both H-intein or intein-L linkage. As opposed to achieving complete efficient cleavage, the protein byproduct of H-intein, intein-L, or triple H-intein-L is produced. However, even in the latter situation, it may be useful to remove the intein component. Thus, strategies have been developed to allow at least partial efficiency of intein cleavage and / or ligation reactions by having tags, preferably internal tags, in the intein.

As described elsewhere herein, in a sample of the culture supernatant from the sORF construct, we found several partially processed intermediates containing intein proteins attached to either the immunoglobulin heavy and / or light chains. Observed. We have designed sORF constructs in which the Pfu lon and Pab lon inteins contain an internal multihistidine tag (IHT). This provides compositions and methods that allow for the rapid and efficient separation of unprocessed contaminants.

We have found that inteins can be modified by inserting peptides or large proteins. Preferably, insertion is made into a solvent accessible loop. By analyzing the sequence alignment of several inteins with structural modeling, we found that solvent accessible loops in both the Pyrococcus abici (PAB) LON intein and Pyrococcus puriosus (PFU) LON intein Confirmed. This loop was located between the endonuclease (H) domain motif and the F / G block of the intein (FIG. 14). FIG. 14 shows certain structural motifs of intein with respect to the location of the preferred location (dashed arrow) of the solvent accessible loop between the H and F / G blocks for the introduction of the insert comprising the tag (see: Information available on the Internet website, http://tools.neb.com/inbase/motifs_endo .php (InBase, The Intein Database: DOD Homing Endonuclease Motifs; InBase Reference: Perler, FB, 2002, InBase, the Intein Database, Nucleic Acids Res. 30, 383-384) (Schematic source showing specific intein structural features, including motifs.) The inventors have found that there are many possible ways in which the regions between the domains shown can be used in the solvent accessible loop. It was determined whether the insertion site was acceptable.According to the above sources, certain features of the conserved residues in Figure 14 appear as follows: boxed amino acids, nucleophiles in standard splicing reactions. Sieve; amino acids conserved in the standard intein, upper characters in the lower case, amino acids in the multimeric intein, which can be spliced by a modified mechanism.

Using site directed mutagenesis, we inserted polyhistidine affinity tags in the loop and tested their ability to function. IHT does not destroy the ability of PAB-LON or PFU-LON intein to self-process and can purify proteins using polyhistidine tags, demonstrating that the loop is solvent accessible. In addition, experiments with the crystal structure of the PFU-RIR1-1 structure suggest that any protein can be inserted into the region as long as they do not substantially or completely disrupt the amino and carboxyl terminal self-catalytic reactions. For example, a polypeptide tag or protein having very close amino and carboxy terminal residues should not substantially destroy the autocatalytic activity of the intein. In a preferred embodiment, a construct is provided having an insert, such as a tag, wherein the construct can exhibit one or more desired intein activities (eg, cleavage and / or linkage). Thus, construct components comprising the corresponding solvent accessible loop of intein can be modified to produce many different functional molecules.

For the Pfu lon intein, tag sequences were inserted. Preferred positions for insertion are where the amino acid sequence at the top of the insertion site is IEFIP (AA 323-327) and the bottom of the insertion site is ISFSP (AA 328-332). In the case of Pab lon intein, tag sequences were inserted. Preferred positions for insertion are where the amino acid sequence at the bottom of the insertion site is IIFDA (AA 291-295) and the bottom of the insertion site is GRLDV (AA 296-300). In each of the Pfu lon and Pab lon intein constructs, the inserted tag sequences included: HHHHHH (SEQ ID NO: 56), HHHHHHHHHH (SEQ ID NO: 57), and HQHQHQ (SEQ ID NO: 58). As evidenced by the latter tag sequence, where H is histidine and Q is glutamine, the insert may be other than a polyhistidine tag. Additional insert sequences can be used as understood in the art.

IHT-modified intein sORF constructs yielded antibody secretion concentrations similar to these without IHT modification. The results indicate that IHT modification not only destroys the ability of intein to function in its self-processing of the sORF product, but also does not prevent correctly processed antibodies from secreting IHT into the medium.

Using fixed metal affinity chromatography, we demonstrated that inteins containing contaminants can be removed quickly and efficiently from Protein A purified antibody preparations via IHT. These IHT constructs enable the inventors to isolate correctly processed antibodies and represent supplemental methods for the production of sORF-derived biologics comprising therapeutic antibodies.

Using internally His-tagged sORF constructs, D2E7 antibody molecules were efficiently separated from the intein-containing protein species in flow through the technique using nickel column chromatography. Similarly, D2E7 antibodies produced using Pab lon HL (−) constructs were also efficiently isolated from intein-containing protein species using Q-columns. IgG samples produced from Pab lon HL (-) purified by Protein A technology, and IgG samples produced from both Pab lon HL (-) / 10 His constructs and purified with both proA and Ni-resins were also subjected to SEC fractionation. Analyzed. The sample after purification showed improved purity of the monomeric IgG species. Size exclusion chromatography (SEC) further removed some remaining contaminants. Purified IgG samples were analyzed for binding affinity for specific antigen, TNFα by BiaCore. The affinity of these samples was indistinguishable from D2E7 antibodies produced using conventional vectors.

Example 8 Pho lon Inteins

The amino acid sequence for the Pho lon intein of Pyrococcus horicosyl OT3 is shown below. See also Inbase, NCBI / Protein according to the NEB intein database, accession number BAA29538.1 in PH0452.

Example  9. Vectors and Light chain Signal Peptide .

Further progress has been made in connection with a single transcription translation frame vector for expression of the protein. In aspects, the vectors use proteins of immunoglobulins for expression in mammalian cells. In such vectors, the structure of the components of the light chain components, including the light chain signal peptide, are designed and generated.

In an embodiment of a single transcription translation frame construct, signal peptides from natural human antibody light chain sources are used. For example, human light chain signal peptides have been reported in V BASE, which includes a database of information in human germline variable region sequences from sources such as Genbank and EMBL data libraries. The V BASE database is affiliated with the MRC Center for Protein Engineering (available via the Internet at http://vbase.mrc-cpe.cam.ac.uk/) for protein processing in Cambridge, UK. [See also, Giudicelli V et al., Nucleic Acids Research, 2006, Vol. 34, Database issue D781-D784; Retter I et al., Nucleic Acids Res. 2005 January 1; 33 (Database Issue): D671-D674]. In particular embodiments, a number of vector designs are provided that can be used to produce IgGl antibodies having various amino acids, including naturally occurring amino acids.

Certain human light chain signal peptides, generally ranging from 19 to 23 amino acids in length, are provided and have the sequences shown in the following table (s) (Hu Vk, human kappa variable region; LCSP, light chain signal peptide). Variant peptides are also provided which may vary in amino acid sequence and / or length in connection with natural peptides comprising such human origin. In the case of provided amino acid sequences, gaps may be indicated for comparison purposes with respect to alignment with one or more other sequences. In an aspect, provided light chain signal peptides or nucleic acid sequences encoding thereto are provided. In one embodiment, an amino acid or nucleic acid sequence is provided as a synthetic construct of an expression vector, a synthesized (as naturally synthesized) molecule, or a sequence such as a recombinant expression product or a segment thereof.

Certain Vkappa signal peptides were substituted with L5, the signal peptide for the immunoglobulin light chain named E7 (corresponding to the light chain of antibody molecule D2E7 with antigen specificity for tumor necrosis factor alpha). In addition, mutation libraries of specific mutants of L5 were constructed and mutant L5 peptides were substituted for native L5 peptides. Mammalian expression vectors were constructed by HL orientation using the Pyrococcus acci lon intein. The following vectors were generated: pTT3-A14-E7-PablonHL, pTT3-A17-E7-PablonHL, pTT3-A18-E7-PablonHL, pTT3-A19-E7-PablonHL, pTT3-A23-E7-PablonHL, pTT3-A26- E7-PablonHL, pTT3-A27-E7-PablonHL, pTT3-B2-E7-PablonHL, pTT3-B3-E7-PablonHL, pTT3-L2-E7-PablonHL, pTT3-L20-E7-PablonHL, pTT3-L25-E7- PablonHL, pTT3-mut-1R-E7-PablonHL, pTT3-mut-1R2G-E7-PablonHL, pTT3-mut-2R-E7-PablonHL, pTT3-mut-2G-E7-PablonHL, pTT3-mut-2R2G-E7- PablonHL, pTT3-mut-3G-E7-PablonHL, pTT3-mut-3R2G-E7-PablonHL, pTT3-mut-4G-E7-PablonHL, pTT3-mut-H + 2G-E7-PablonHL. These constructs were prepared on a PTT3 vector backbone. The vector has an EBV replication origin that allows episomal amplification thereof in 293E cells (cells expressing Epstein-Barr virus nuclear antigen 1) transfected in suspension culture. Each vector had one ORF, driven by a CMV promoter. In the ORF, the intein sequence was inserted in the frame between the antibody heavy and light chains in the order of HC-intein-LC. A schematic of the construct structure for pTT3-A18-E7-PablonHL is shown in FIG. 15.

The construct was introduced into 293E cells via transient transfection and a number of transient expression experiments were performed. In the experiments provided, samples were collected and analyzed from the supernatants of cultures of transfected cells 8 days after transfection. Samples containing the concentration of antibody secreted as assessed by IgG ELISA are shown in the table below with micrograms of antibody per ml of sample. The natural control was the vector used for the L5 LCSP sequence. As another control (not shown in the table), two conventional vector systems expressing the same antibody and using the same regulatory components were included in these experiments; The antibody secretion concentration produced from this control vector system was 80-206 μg / ml. IgG secretion concentrations produced by some of the sORF construct designs using these light chain signal peptides are comparable in the order of range produced using conventional vectors. These expression levels are significantly higher than those using native L5 E7 signal peptide (2.0 μg / ml using the Fablon HL (+) construct). These antibody secretion concentrations are also significantly higher than those produced using the "2A" technique reported to be 1.6 μg / ml in mammalian cells (Fang et al., 2005, Nature Biotechnology 23: 584- 590).

Of the specified constructs for which antibody product concentrations were measured, products from five of the constructs that produced antibodies secreted at maximum concentration were selected for further analysis. The product corresponded to the following five constructs: pTT3-A17-E7-PablonHL, pTT3-A18-E7-PablonHL, pTT3-A19-E7-PablonHL, pTT3-A26-E7-PablonHL, and pTT3-mutH + 2G- E7-PablonHL. Secreted antibodies produced from these constructs were purified by Protein A affinity chromatography and analyzed on a reducing SDS-PAGE gel and the N-terminal amino acid sequences for their HC and LC were determined. Samples produced using pTT3-A18-E7-PablonHL contained protein transfer bands corresponding to the antibody heavy and light chains in a shift indistinguishable from similar antibodies produced by conventional methods. On the reducing gel, in addition to the bands corresponding to the antibodies HC and LC, there were also two high molecular weight bands which appeared to correspond to the unprocessed 3-part protein (HC-intein-LC). Such construct-related contaminants, such as unprocessed or partially processed proteins, can be conveniently removed in accordance with conventional techniques as described herein. Reference is made to FIG. 16 which shows an example of the results from the SDS-PAGE analysis. Secreted IgG antibodies were purified by Protein A affinity chromatography and separated using a technique of SDS-PAGE in a gel under reducing conditions. Samples in lanes from left to right are as follows: Lane (1) SeeBlue Plus2 protein standard [manufacturer; Invitrogen] protein molecular weight markers; (2) control, D2E7 antibody produced with a traditional non-sORF expression vector system; (3) Pab-lon HL (−); (4) pTT3-A18-E7-PablonHL.

Intracellular samples of the product of the expression construct were also analyzed by Western blot analysis using antibodies against both HC and LC. Similar protein species were observed as in cultured supernatants. The N-terminal amino acid sequences of both heavy and light chains were determined to be natural by mass spectrometry analysis. The analysis confirmed that the A18 signal peptide cleavage occurred precisely at the exact point desired for expression of the light chain. In addition, similarity to traditionally expressed D2E7 was demonstrated using cation exchange chromatography (CIEX), which separates proteins based on total surface charge and can detect variants and impurities of D2E7. Thus, A18 signal peptides can be used in sORF vectors for antibody expression and can efficiently express fully processed and assembled antibody products.

In addition to the transient expression systems described above, stable cell lines were also generated for expression of antibody products. Stable CHO cell lines were prepared as sORF expression constructs using vectors with A18 light chain signal peptide components. sORF construct A18-E7-PablonHL was cloned into plasmid pA190 using recombinant technology. See FIG. 18 which provides a schematic of the construct structure for pBJ-A18-LC-Pablon-HL (also referred to as pA190-A18-E7-PablonHL). The constructs were transfected into CHO B3.2 cells by the calcium phosphate method and plated into 48 96-well plates in minimal essential medium (MEM) alpha medium containing 5% FBS. Transfection samples were screened and subjected to amplification methods with MTX of 100 nM or less. The expression results of the constructs in the cultures were characterized. At 100 nM MTX, cells with construct pA190-A18-E7-PablonHL expressed antibodies ranging from 1.1 to 16.9 μg / ml in samples of the culture supernatant. Four maximal expressing clones were cloned at the limiting dilution. Subclone was tested and found to express the antibody in an amount of 2.9 to 31.8 μg / ml. Four of the cloned cell lines with maximum expression levels were adapted to grow in suspension and produced an average amount of 31-44 μg / ml as measured from samples collected on day 4 of culture.

The ability of the construct to produce mature light chain product was evaluated. See FIG. 17, which provides results from Western blot experiments. Samples of intracellular antibody products were characterized. Whole cell lysates are separated according to SDS-PAGE in the gel under reducing conditions, transferred to nitrocellulose membranes, blocked with blocking solution (skim milk powder in TTBS, Tris-buffered saline containing Tween 20), heavy chain or It was incubated with horseradish peroxidase-conjugated antibody to the light chain and developed using ECL (enhanced chemiluminescent) reagent. Samples according to in-lane construct designation from left to right in the blot are: uncounted lanes on the left, molecular weight markers; (Lane 1) control, D2E7 from CHO cells; (2) control, transient transfection with HEK293 cells, pTT3-A18-E7-PablonHL; (3-11) various clones from pA190-A18-E7-PablonHL corresponding to clone numbers 1, 3, 7, 9, 12, 14, 18, 15, and 13, respectively. Arrows labeled with the letters “a” and “b” indicate expression products as follows: (a) upper band, light chain with signal peptide, and (b) lower band, mature light chain. In mature light chain products, the signal peptide was cleaved to produce a product with a lower molecular weight associated with the precursor. The results demonstrate that the A18 light chain signal peptide component can express and produce a fully mature light chain product comparable to that of the D2E7 antibody.

References and explanations for incorporation by variation

Any sequence listing information is considered part of this specification.

All references cited throughout this specification, such as issued or registered patents or equivalents; Patent application publications; Patent documents, including unpublished patent applications; And non-patent literature documents or other feedstocks are incorporated herein by reference in their entirety, through individual incorporation by reference. In the event of any inconsistency between the cited documents and the disclosure herein, the disclosure herein takes precedence. Some references provided herein provide information, such as, for example, a source of starting material, additional starting materials, additional reagents, additional synthetic methods, additional analytical methods, additional biological materials, additional cells, and additional inventions. It is incorporated by reference to provide details regarding its use.

All patents and publications mentioned herein are indicative of the level of skill in the art to which this invention belongs. The references cited herein refer to the art as their publication date or application date, and the information is intended to be used to exclude special aspects belonging to the prior art if necessary. For example, where the composition of a substance is claimed herein, compounds known and available in the art prior to the applicant's invention, such as those provided in the references for which the disclosure is cited herein, are those It is not intended to be included in the composition of.

All attachments or attachments to the application are incorporated by reference as part of the specification and / or drawings.

When a compound, construct or composition is claimed, it is to be understood that the compound, construct and composition known in the art, including those taught in the references described herein, is not intended to be included. Where a Markush group or other group is used herein, all individual members of the group and all possible combinations and subcombinations from within the group are also set individually and are intended to be included in the description.

When the terms “comprise, comprise”, “included”, or “comprising” are used herein, they refer to the presence or addition of one or more other features, integers, steps, components or groups thereof. It should be construed as indicating the presence of the described features, integers, steps or components without excluding them. Thus, as used herein, 'comprising' is synonymous with 'comprising', 'containing', 'having' or 'characterized by' and is comprehensive or extensible and further, not listed components or It does not exclude method steps. As used herein, “consisting of” excludes any ingredient, step or ingredient not specified in the claims. As used herein, “consisting essentially of” does not exclude substances or steps that do not substantially affect the basic and novel features of the claims (eg, related to the active ingredient). In each of the examples herein, any of the terms “comprising”, “consisting essentially of” and “consisting of” consists of distinct aspects and / or regions that do not necessarily coexist by being replaced by at least two other terms. Can be. Aspects of the invention described herein descriptively may be suitably practiced in the absence of any component or ingredients or limitations or limitations specifically described herein.

Whenever a range, eg, a temperature range, a time range, a composition or concentration range, or other value range, is described herein, all individual values included in the provided ranges, as well as all intermediate and subranges, are It is intended to be included in the description. The present invention is not limited in the manner of examples or descriptions and is not limited by the described aspects, including any shown, illustrated in the drawings or illustrated in the specification. It is to be understood that any subrange or individual value in the range or subrange included in the description herein may be excluded from the claims herein.

The present invention has been described with reference to various specific and / or preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made within the spirit and scope of the invention. To those skilled in the art, compositions, methods, devices, device components, materials, processes and techniques other than those specifically described herein are described broadly herein without reliance on undue experimentation. Can be used in the practice of the present invention; It will be apparent that this may extend to, for example, starting materials, biological materials, reagents, synthetic methods, purification methods, analytical methods, assay methods and biological methods other than those specifically exemplified. All previous functional equivalents known in the art described herein (eg, compositions, methods, devices, device components, materials, processes and techniques, etc.) are intended to be included in the present invention. The terms and expressions used are used in terms of technology and are not intended to be limiting, and are not intended to be used with the terms and expressions other than any equivalent or part of the features shown and described, but various modifications of the invention claimed It is recognized that it is possible within the domain. Thus, although the invention is specifically described by its aspects, preferred embodiments, any feature, modification, and change of the concepts described herein may be reclassified by those skilled in the art and such modifications and changes Should be considered to be within the scope of the present invention as defined by the appended claims.

References

The present application is specifically cited by reference in its entirety in each of the following items:

                         SEQUENCE LISTING <110> ABBOTT LABORATORIES <120> SORF CONSTRUCTS AND MULTIPLE GENE EXPRESSION <130> 54-08 WO <150> US 61 / 256,544 <151> 2009-10-30 <160> 108 <170> Kopatentin 1.71 <210> 1 <211> 335 <212> PRT Pyrococcus abyssi <400> 1 Gln Cys Phe Ser Gly Glu Glu Thr Val Val Ile Arg Glu Asn Gly Glu 1 5 10 15 Val Lys Val Leu Arg Leu Lys Asp Phe Val Glu Lys Ala Leu Glu Lys             20 25 30 Pro Ser Gly Glu Gly Leu Asp Gly Asp Val Lys Val Val Tyr His Asp         35 40 45 Phe Arg Asn Glu Asn Val Glu Val Leu Thr Lys Asp Gly Phe Thr Lys     50 55 60 Leu Leu Tyr Ala Asn Lys Arg Ile Gly Lys Gln Lys Leu Arg Arg Val 65 70 75 80 Val Asn Leu Glu Lys Asp Tyr Trp Phe Ala Leu Thr Pro Asp His Lys                 85 90 95 Val Tyr Thr Thr Asp Gly Leu Lys Glu Ala Gly Glu Ile Thr Glu Lys             100 105 110 Asp Glu Leu Ile Ser Val Pro Ile Thr Val Phe Asp Cys Glu Asp Glu         115 120 125 Asp Leu Lys Lys Ile Gly Leu Leu Pro Leu Thr Ser Asp Asp Glu Arg     130 135 140 Leu Arg Lys Ile Ala Thr Leu Met Gly Ile Leu Phe Asn Gly Gly Ser 145 150 155 160 Ile Asp Glu Gly Leu Gly Val Leu Thr Leu Lys Ser Glu Arg Ser Val                 165 170 175 Ile Glu Lys Phe Val Ile Thr Leu Lys Glu Leu Phe Gly Lys Phe Glu             180 185 190 Tyr Glu Ile Ile Lys Glu Glu Asn Thr Ile Leu Lys Thr Arg Asp Pro         195 200 205 Arg Ile Ile Lys Phe Leu Val Gly Leu Gly Ala Pro Ile Glu Gly Lys     210 215 220 Asp Leu Lys Met Pro Trp Trp Val Lys Leu Lys Pro Ser Leu Phe Leu 225 230 235 240 Ala Phe Leu Glu Gly Phe Arg Ala His Ile Val Glu Gln Leu Val Asp                 245 250 255 Asp Pro Asn Lys Asn Leu Pro Phe Phe Gln Glu Leu Ser Trp Tyr Leu             260 265 270 Gly Leu Phe Gly Ile Lys Ala Asp Ile Lys Val Glu Glu Val Gly Asp         275 280 285 Lys His Lys Ile Ile Phe Asp Ala Gly Arg Leu Asp Val Asp Lys Gln     290 295 300 Phe Ile Glu Thr Trp Glu Asp Val Glu Val Thr Tyr Asn Leu Thr Thr 305 310 315 320 Glu Lys Gly Asn Leu Leu Ala Asn Gly Leu Phe Val Lys Asn Ser                 325 330 335 <210> 2 <211> 999 <212> DNA Pyrococcus abyssi <400> 2 tgcttcagcg gcgaggaaac cgtggtgatc cgggagaacg gcgaggtgaa ggtgctgcgg 60 ctgaaggact tcgtggagaa ggccctggaa aagccctccg gcgagggcct ggacggcgac 120 gtgaaagtgg tgtaccacga cttccggaac gagaacgtgg aggtgctgac caaggacggc 180 ttcaccaagc tgctgtacgc caacaagcgg atcggcaagc agaaactgcg gcgggtggtg 240 aacctggaaa aggactactg gttcgccctg acccccgacc acaaggtgta caccaccgac 300 ggcctgaaag aggccggcga gatcaccgag aaggacgagc tgatcagcgt gcccatcacc 360 gtgttcgact gcgaggacga ggacctgaag aagatcggcc tgctgcccct gaccagcgac 420 gacgagcggc tgcggaagat cgccaccctg atgggcatcc tgttcaacgg cggcagcatc 480 gatgagggcc tgggcgtgct gaccctgaag agcgagcgga gcgtgatcga gaagttcgtg 540 atcaccctga aagagctgtt cggcaagttc gagtacgaga tcatcaaaga ggaaaacacc 600 atcctgaaaa cccgggaccc ccggatcatc aagtttctgg tgggcctggg agcccccatc 660 gagggcaagg atctgaagat gccttggtgg gtgaagctga agcccagcct gttcctggcc 720 ttcctggaag gcttccgggc ccacatcgtg gagcagctgg tcgacgaccc caacaagaat 780 ctgcccttct ttcaggaact gagctggtat ctgggcctgt tcggcatcaa ggccgacatc 840 aaggtggagg aagtgggcga caagcacaag atcatcttcg acgccggcag gctggacgtg 900 gacaagcagt tcatcgagac ctgggaggat gtggaggtga cctacaacct gaccacagag 960 aagggcaatc tgctggccaa cggcctgttc gtgaagaac 999 <210> 3 <211> 333 <212> PRT Pyrococcus abyssi <400> 3 Cys Phe Ser Gly Glu Glu Thr Val Val Ile Arg Glu Asn Gly Glu Val 1 5 10 15 Lys Val Leu Arg Leu Lys Asp Phe Val Glu Lys Ala Leu Glu Lys Pro             20 25 30 Ser Gly Glu Gly Leu Asp Gly Asp Val Lys Val Val Tyr His Asp Phe         35 40 45 Arg Asn Glu Asn Val Glu Val Leu Thr Lys Asp Gly Phe Thr Lys Leu     50 55 60 Leu Tyr Ala Asn Lys Arg Ile Gly Lys Gln Lys Leu Arg Arg Val Val 65 70 75 80 Asn Leu Glu Lys Asp Tyr Trp Phe Ala Leu Thr Pro Asp His Lys Val                 85 90 95 Tyr Thr Thr Asp Gly Leu Lys Glu Ala Gly Glu Ile Thr Glu Lys Asp             100 105 110 Glu Leu Ile Ser Val Pro Ile Thr Val Phe Asp Cys Glu Asp Glu Asp         115 120 125 Leu Lys Lys Ile Gly Leu Leu Pro Leu Thr Ser Asp Asp Glu Arg Leu     130 135 140 Arg Lys Ile Ala Thr Leu Met Gly Ile Leu Phe Asn Gly Ser Ile 145 150 155 160 Asp Glu Gly Leu Gly Val Leu Thr Leu Lys Ser Glu Arg Ser Val Ile                 165 170 175 Glu Lys Phe Val Ile Thr Leu Lys Glu Leu Phe Gly Lys Phe Glu Tyr             180 185 190 Glu Ile Ile Lys Glu Glu Asn Thr Ile Leu Lys Thr Arg Asp Pro Arg         195 200 205 Ile Ile Lys Phe Leu Val Gly Leu Gly Ala Pro Ile Glu Gly Lys Asp     210 215 220 Leu Lys Met Pro Trp Trp Val Lys Leu Lys Pro Ser Leu Phe Leu Ala 225 230 235 240 Phe Leu Glu Gly Phe Arg Ala His Ile Val Glu Gln Leu Val Asp Asp                 245 250 255 Pro Asn Lys Asn Leu Pro Phe Phe Gln Glu Leu Ser Trp Tyr Leu Gly             260 265 270 Leu Phe Gly Ile Lys Ala Asp Ile Lys Val Glu Glu Val Gly Asp Lys         275 280 285 His Lys Ile Ile Phe Asp Ala Gly Arg Leu Asp Val Asp Lys Gln Phe     290 295 300 Ile Glu Thr Trp Glu Asp Val Glu Val Thr Tyr Asn Leu Thr Thr Glu 305 310 315 320 Lys Gly Asn Leu Leu Ala Asn Gly Leu Phe Val Lys Asn                 325 330 <210> 4 <211> 403 <212> PRT <213> Pyrococcus furiosus <400> 4 Gln Cys Phe Ser Gly Glu Glu Val Ile Leu Ile Glu Lys Asp Gly Glu 1 5 10 15 Lys Lys Val Phe Lys Leu Arg Glu Phe Val Asp Gly Leu Leu Lys Glu             20 25 30 Ala Ser Gly Glu Gly Met Asp Gly Ser Ile Arg Val Val Tyr Lys Asp         35 40 45 Leu Gln Gly Glu Asn Ile Lys Ile Leu Thr Lys Asp Gly Leu Val Lys     50 55 60 Leu Leu Tyr Val Asn Arg Arg Glu Gly Lys Gln Lys Leu Arg Lys Ile 65 70 75 80 Val Asn Leu Glu Lys Asp Tyr Trp Leu Ala Leu Thr Pro Glu His Lys                 85 90 95 Val Tyr Thr Ile Lys Gly Leu Lys Glu Ala Gly Glu Ile Thr Lys Asp             100 105 110 Asp Glu Ile Ile Arg Val Pro Leu Thr Ile Leu Asp Gly Phe Asp Val         115 120 125 Ala Glu Lys Ser Ile Arg Glu Glu Leu Glu Arg Leu Ser Leu Leu Pro     130 135 140 Leu Asn Ser Glu Asp Ser Arg Leu Glu Lys Ile Ala Gly Ile Met Gly 145 150 155 160 Ala Leu Phe Gly Ser Gly Gly Ile Asp Glu Asn Leu Asn Thr Leu Ser                 165 170 175 Phe Val Ser Ser Glu Lys Lys Thr Ile Glu Gln Phe Val Lys Ala Leu             180 185 190 Ser Glu Leu Phe Gly Glu Phe Asp Tyr Lys Ile Glu Glu Lys Glu Asn         195 200 205 Ser Ile Ile Phe Arg Thr Cys Asp Lys Arg Ile Val Thr Phe Phe Ala     210 215 220 Thr Leu Gly Ala Pro Val Gly Asp Lys Ser Lys Val Lys Leu Lys Leu 225 230 235 240 Pro Trp Trp Val Lys Leu Lys Pro Ser Leu Phe Leu Ala Phe Met Asp                 245 250 255 Gly Leu Tyr Ser Ser Asn Arg Asn Asp Lys Glu Ile Leu Glu Ile Thr             260 265 270 Gln Leu Thr Asp Asn Val Glu Thr Phe Phe Glu Glu Ile Ser Trp Tyr         275 280 285 Leu Ser Phe Phe Gly Ile Lys Ala Glu Ala Glu Glu Asp Glu Glu Lys     290 295 300 Asp Lys Tyr Arg Ala Arg Leu Thr Leu Ser Ser Ser Ile Asp Asn Met 305 310 315 320 Leu Asn Phe Ile Glu Phe Ile Pro Ile Ser Phe Ser Pro Ala Lys Arg                 325 330 335 Glu Lys Phe Phe Lys Glu Ile Glu Lys Tyr Leu Glu Tyr Ser Ile Pro             340 345 350 Glu Lys Thr Glu Asp Leu Lys Lys Arg Val Lys Arg Val Lys Lys Gly         355 360 365 Glu Arg Arg Asn Phe Leu Glu Ser Trp Glu Glu Val Glu Val Thr Tyr     370 375 380 Asn Val Thr Thr Glu Thr Gly Asn Leu Leu Ala Asn Gly Leu Phe Val 385 390 395 400 Lys asn ser              <210> 5 <211> 1203 <212> DNA <213> Pyrococcus furiosus <400> 5 tgttttagcg gtgaagaagt tatcttaatt gaaaaggacg gagagaaaaa agtcttcaaa 60 cttagggagt tcgttgacgg tctccttaag gaggcgtctg gagaagggat ggacggaagt 120 attagagtag tttataaaga tcttcaaggg gaaaacataa aaatactcac aaaagacgga 180 cttgtaaagc tcctttatgt caatagaaga gaagggaagc aaaagcttag aaaaatagta 240 aatcttgaaa aggattattg gcttgcatta acacctgaac ataaagtgta cacaataaag 300 ggccttaaag aagctggaga gataactaaa gatgatgaga taataagagt gcctctcaca 360 attcttgacg gctttgacgt agccgagaag agtataagag aggaacttga aaggcttagc 420 ctacttccac taaatagtga agacagtaga ctagaaaaga tagcaggaat catgggcgca 480 ctctttggta gtggaggtat cgatgagaat ctcaataccc ttagctttgt ttctagcgag 540 aagaaaacaa ttgaacagtt tgttaaagca ctcagcgagc tcttcgggga atttgactat 600 aaaattgaag aaaaagaaaa cagcattatt ttcagaacat gtgataaaag aatagtgacc 660 ttctttgcta cacttggtgc accagttgga gacaaaagca aagttaagct taagcttcca 720 tggtgggtca agcttaagcc gtcacttttc ctcgccttca tggatggtct ctacagtagc 780 aataggaatg acaaagaaat cctcgaaata actcaactta ctgacaacgt cgaaacgttc 840 ttcgaggaaa tatcttggta tctgagcttc tttggaatta aggcagaagc tgaagaggat 900 gaagaaaaag ataaatacag ggctagactt acgctatcct catcaataga caacatgctt 960 aatttcattg agttcattcc aataagcttt tctccagcaa agagagaaaa attctttaag 1020 gaaattgaaa aatatctgga atatagcatt cccgaaaaga ctgaggatct taagaaacga 1080 gttaagagag ttaagaaggg agagagaagg aatttcctcg aaagctggga ggaagttgaa 1140 gttacttaca acgtaactac agagacagga aatctacttg ctaacggtct atttgttaag 1200 aac 1203 <210> 6 <211> 401 <212> PRT <213> Pyrococcus furiosus <400> 6 Cys Phe Ser Gly Glu Glu Val Ile Leu Ile Glu Lys Asp Gly Glu Lys 1 5 10 15 Lys Val Phe Lys Leu Arg Glu Phe Val Asp Gly Leu Leu Lys Glu Ala             20 25 30 Ser Gly Glu Gly Met Asp Gly Ser Ile Arg Val Val Tyr Lys Asp Leu         35 40 45 Gln Gly Glu Asn Ile Lys Ile Leu Thr Lys Asp Gly Leu Val Lys Leu     50 55 60 Leu Tyr Val Asn Arg Arg Glu Gly Lys Gln Lys Leu Arg Lys Ile Val 65 70 75 80 Asn Leu Glu Lys Asp Tyr Trp Leu Ala Leu Thr Pro Glu His Lys Val                 85 90 95 Tyr Thr Ile Lys Gly Leu Lys Glu Ala Gly Glu Ile Thr Lys Asp Asp             100 105 110 Glu Ile Ile Arg Val Pro Leu Thr Ile Leu Asp Gly Phe Asp Val Ala         115 120 125 Glu Lys Ser Ile Arg Glu Glu Leu Glu Arg Leu Ser Leu Leu Pro Leu     130 135 140 Asn Ser Glu Asp Ser Arg Leu Glu Lys Ile Ala Gly Ile Met Gly Ala 145 150 155 160 Leu Phe Gly Ser Gly Gly Ile Asp Glu Asn Leu Asn Thr Leu Ser Phe                 165 170 175 Val Ser Ser Glu Lys Lys Thr Ile Glu Gln Phe Val Lys Ala Leu Ser             180 185 190 Glu Leu Phe Gly Glu Phe Asp Tyr Lys Ile Glu Glu Lys Glu Asn Ser         195 200 205 Ile Ile Phe Arg Thr Cys Asp Lys Arg Ile Val Thr Phe Phe Ala Thr     210 215 220 Leu Gly Ala Pro Val Gly Asp Lys Ser Lys Val Lys Leu Lys Leu Pro 225 230 235 240 Trp Trp Val Lys Leu Lys Pro Ser Leu Phe Leu Ala Phe Met Asp Gly                 245 250 255 Leu Tyr Ser Ser Asn Arg Asn Asp Lys Glu Ile Leu Glu Ile Thr Gln             260 265 270 Leu Thr Asp Asn Val Glu Thr Phe Phe Glu Glu Ile Ser Trp Tyr Leu         275 280 285 Ser Phe Phe Gly Ile Lys Ala Glu Ala Glu Glu Asp Glu Glu Lys Asp     290 295 300 Lys Tyr Arg Ala Arg Leu Thr Leu Ser Ser Ser Ile Asp Asn Met Leu 305 310 315 320 Asn Phe Ile Glu Phe Ile Pro Ile Ser Phe Ser Pro Ala Lys Arg Glu                 325 330 335 Lys Phe Phe Lys Glu Ile Glu Lys Tyr Leu Glu Tyr Ser Ile Pro Glu             340 345 350 Lys Thr Glu Asp Leu Lys Lys Arg Val Lys Arg Val Lys Lys Gly Glu         355 360 365 Arg Arg Asn Phe Leu Glu Ser Trp Glu Glu Val Glu Val Thr Tyr Asn     370 375 380 Val Thr Thr Glu Thr Gly Asn Leu Leu Ala Asn Gly Leu Phe Val Lys 385 390 395 400 Asn      <210> 7 <211> 333 <212> PRT Pyrococcus abyssi <400> 7 Cys Phe Ser Gly Glu Glu Thr Val Val Ile Arg Glu Asn Gly Glu Val 1 5 10 15 Lys Val Leu Arg Leu Lys Asp Phe Val Glu Lys Ala Leu Glu Lys Pro             20 25 30 Ser Gly Glu Gly Leu Asp Gly Asp Val Lys Val Val Tyr His Asp Phe         35 40 45 Arg Asn Glu Asn Val Glu Val Leu Thr Lys Asp Gly Phe Thr Lys Leu     50 55 60 Leu Tyr Ala Asn Lys Arg Ile Gly Lys Gln Lys Leu Arg Arg Val Val 65 70 75 80 Asn Leu Glu Lys Asp Tyr Trp Phe Ala Leu Thr Pro Asp His Lys Val                 85 90 95 Tyr Thr Thr Asp Gly Leu Lys Glu Ala Gly Glu Ile Thr Glu Lys Asp             100 105 110 Glu Leu Ile Ser Val Pro Ile Thr Val Phe Asp Cys Glu Asp Glu Asp         115 120 125 Leu Lys Lys Ile Gly Leu Leu Pro Leu Thr Ser Asp Asp Glu Arg Leu     130 135 140 Arg Lys Ile Ala Thr Leu Met Gly Ile Leu Phe Asn Gly Ser Ile 145 150 155 160 Asp Glu Gly Leu Gly Val Leu Thr Leu Lys Ser Glu Arg Ser Val Ile                 165 170 175 Glu Lys Phe Val Ile Thr Leu Lys Glu Leu Phe Gly Lys Phe Glu Tyr             180 185 190 Glu Ile Ile Lys Glu Glu Asn Thr Ile Leu Lys Thr Arg Asp Pro Arg         195 200 205 Ile Ile Lys Phe Leu Val Gly Leu Gly Ala Pro Ile Glu Gly Lys Asp     210 215 220 Leu Lys Met Pro Trp Trp Val Lys Leu Lys Pro Ser Leu Phe Leu Ala 225 230 235 240 Phe Leu Glu Gly Phe Arg Ala His Ile Val Glu Gln Leu Val Asp Asp                 245 250 255 Pro Asn Lys Asn Leu Pro Phe Phe Gln Glu Leu Ser Trp Tyr Leu Gly             260 265 270 Leu Phe Gly Ile Lys Ala Asp Ile Lys Val Glu Glu Val Gly Asp Lys         275 280 285 His Lys Ile Ile Phe Asp Ala Gly Arg Leu Asp Val Asp Lys Gln Phe     290 295 300 Ile Glu Thr Trp Glu Asp Val Glu Val Thr Tyr Asn Leu Thr Thr Glu 305 310 315 320 Lys Gly Asn Leu Leu Ala Asn Gly Leu Phe Val Lys Asn                 325 330 <210> 8 <211> 999 <212> DNA Pyrococcus abyssi <400> 8 tgcttcagcg gcgaggaaac cgtggtgatc cgggagaacg gcgaggtgaa ggtgctgcgg 60 ctgaaggact tcgtggagaa ggccctggaa aagccctccg gcgagggcct ggacggcgac 120 gtgaaagtgg tgtaccacga cttccggaac gagaacgtgg aggtgctgac caaggacggc 180 ttcaccaagc tgctgtacgc caacaagcgg atcggcaagc agaaactgcg gcgggtggtg 240 aacctggaaa aggactactg gttcgccctg acccccgacc acaaggtgta caccaccgac 300 ggcctgaaag aggccggcga gatcaccgag aaggacgagc tgatcagcgt gcccatcacc 360 gtgttcgact gcgaggacga ggacctgaag aagatcggcc tgctgcccct gaccagcgac 420 gacgagcggc tgcggaagat cgccaccctg atgggcatcc tgttcaacgg cggcagcatc 480 gatgagggcc tgggcgtgct gaccctgaag agcgagcgga gcgtgatcga gaagttcgtg 540 atcaccctga aagagctgtt cggcaagttc gagtacgaga tcatcaaaga ggaaaacacc 600 atcctgaaaa cccgggaccc ccggatcatc aagtttctgg tgggcctggg agcccccatc 660 gagggcaagg atctgaagat gccttggtgg gtgaagctga agcccagcct gttcctggcc 720 ttcctggaag gcttccgggc ccacatcgtg gagcagctgg tcgacgaccc caacaagaat 780 ctgcccttct ttcaggaact gagctggtat ctgggcctgt tcggcatcaa ggccgacatc 840 aaggtggagg aagtgggcga caagcacaag atcatcttcg acgccggcag gctggacgtg 900 gacaagcagt tcatcgagac ctgggaggat gtggaggtga cctacaacct gaccacagag 960 aagggcaatc tgctggccaa cggcctgttc gtgaagaac 999 <210> 9 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 9 Glu Val Gln Leu Val Glu Ser Gly Gly Gly 1 5 10 <210> 10 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 10 Met Glu Val Gln Leu Val Glu Ser Gly Gly 1 5 10 <210> 11 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 11 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 1 5 10 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 12 Met Asp Ile Gln Met Thr Gln Ser Pro Ser 1 5 10 <210> 13 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 13 Ala Asn Gly Leu Phe Val Lys Asn 1 5 <210> 14 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 14 Met Arg Ala Lys Arg 1 5 <210> 15 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 15 His Ala Arg Gly Val Phe Arg Arg 1 5 <210> 16 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 16 Met Asp Arg Gly Val Phe Arg Arg 1 5 <210> 17 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 17 Asp Ile Gln Met Thr Gln Ser 1 5 <210> 18 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 18 Ala Ile Gln Met Thr Gln Ser 1 5 <210> 19 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 19 Asn Ile Gln Met Thr Gln Ser 1 5 <210> 20 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 20 Asn Phe Gln Met Thr Gln Ser 1 5 <210> 21 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 21 Met Asp Ile Gln Met Thr Gln Ser 1 5 <210> 22 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 22 Met Arg Ala Lys Arg Asp Ile Gln Met Thr Gln Ser 1 5 10 <210> 23 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 23 Tyr Pro Asp Ile Gln Met Thr Gln Ser 1 5 <210> 24 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 24 Arg Pro Asp Ile Gln Met Thr Gln Ser 1 5 <210> 25 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 25 Val Pro Asp Ile Gln Met Thr Gln Ser 1 5 <210> 26 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 26 Gln Pro Asp Ile Gln Met Thr Gln Ser 1 5 <210> 27 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 27 Ala Pro Asp Ile Gln Met Thr Gln Ser 1 5 <210> 28 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 28 His Ala Asp Ile Gln Met Thr Gln Ser 1 5 <210> 29 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 29 Tyr Ala Asp Ile Gln Met Thr Gln Ser 1 5 <210> 30 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 30 Met Pro Asp Ile Gln Met Thr Gln Ser 1 5 <210> 31 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 31 Met Ala Asp Ile Gln Met Thr Gln Ser 1 5 <210> 32 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 32 His Ala Arg Gly Val Phe Arg Arg Asp Ile Gln Met Thr Gln Ser 1 5 10 15 <210> 33 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 33 Met Asp Arg Gly Val Phe Arg Arg Asp Ile Gln Met Thr Gln Ser 1 5 10 15 <210> 34 <211> 504 <212> DNA <213> Methanococcus jannaschii <400> 34 gctctggcct acgacgagcc catctacctg agcgacggca acatcatcaa catcggcgag 60 ttcgtggaca agttcttcaa gaagtacaag aacagcatca agaaagagga caacggcttc 120 ggctggatcg acatcggcaa cgagaacatc tacatcaaga gcttcaacaa gctgtccctg 180 atcatcgagg acaagcggat cctgagagtg tggcggaaga agtacagcgg caagctgatc 240 aagatcacca ccaagaaccg gcgggagatc accctgaccc acgaccaccc cgtgtacatc 300 agcaagaccg gcgaggtgct ggaaatcaac gccgagatgg tgaaagtggg cgactacatc 360 tatatcccca agaacaacac catcaacctg gacgaggtga tcaaggtgga gaccgtggac 420 tacaacggcc acatctacga cctgaccgtg gaggacaacc acacctacat cgccggcaag 480 aacgagggct tcgccgtgag caac 504 <210> 35 <211> 168 <212> PRT <213> Methanococcus jannaschii <400> 35 Ala Leu Ala Tyr Asp Glu Pro Ile Tyr Leu Ser Asp Gly Asn Ile Ile 1 5 10 15 Asn Ile Gly Glu Phe Val Asp Lys Phe Phe Lys Lys Tyr Lys Asn Ser             20 25 30 Ile Lys Lys Glu Asp Asn Gly Phe Gly Trp Ile Asp Ile Gly Asn Glu         35 40 45 Asn Ile Tyr Ile Lys Ser Phe Asn Lys Leu Ser Leu Ile Ile Glu Asp     50 55 60 Lys Arg Ile Leu Arg Val Trp Arg Lys Lys Tyr Ser Gly Lys Leu Ile 65 70 75 80 Lys Ile Thr Thr Lys Asn Arg Arg Glu Ile Thr Leu Thr His Asp His                 85 90 95 Pro Val Tyr Ile Ser Lys Thr Gly Glu Val Leu Glu Ile Asn Ala Glu             100 105 110 Met Val Lys Val Gly Asp Tyr Ile Tyr Ile Pro Lys Asn Asn Thr Ile         115 120 125 Asn Leu Asp Glu Val Ile Lys Val Glu Thr Val Asp Tyr Asn Gly His     130 135 140 Ile Tyr Asp Leu Thr Val Glu Asp Asn His Thr Tyr Ile Ala Gly Lys 145 150 155 160 Asn Glu Gly Phe Ala Val Ser Asn                 165 <210> 36 <211> 588 <212> DNA Pyrococcus abyssi <400> 36 gctctgtact acttcagcga gatccagctg cccaacggca aagagttcat cggcaaactg 60 gtggacgagc tgttcgagaa gtaccacgac aagatcggca agtacaagga catggaatac 120 gtggagctga acgaagagga caccttcgag gtgatcagca tcggccccga cctgagcgcc 180 aggcggcaca aggtgaccca cgtgtggcgg cggaaggtga aagacggcga gaagctggtg 240 aagatccgga ccgccagcgg caaagaactg gtgctgaccc aggaccaccc cgtgttcgtg 300 ctgctgggcc gggacgtggc cagacgggac gccggcaacg tgaaagtggg cgacgagatc 360 gccgtgctga acaccaggcc cgacttcagc gtgctgtccc cccctgccat gcccgagctg 420 ctgtccgagc ccttcaacta cgagctgtcc agcatcggcg acgtggcctg ggacgaggtg 480 gtggaggtgg acgagatcga cgccaagggc ctgggcgtgg agtacctgta cgacctgacc 540 gtggacatca accacaacta cgtggccaac ggcatcgtgg tgtccaac 588 <210> 37 <400> 37 000 <210> 38 <211> 1566 <212> DNA <213> Pyrococcus furiosus <400> 38 gcactttacg atttctctgt catccaacta tctaatggta gatttgtact tataggagat 60 ttagtcgagg aattattcaa gaagtatgcc gagaaaatta aaacatacaa agaccttgag 120 tacatagagc ttaacgagga agaccgtttt gaagttgtta gtgttagtcc agatttgaag 180 gctaataaac atgttgtctc aagagtttgg agaagaaagg tcagagaggg ggaaaagcta 240 atacgcataa agacgagaac tggcaacgaa ataatcctca ctagaaatca tccgctattt 300 gccttctcca atggagacgt agtcagaaaa gaggccgaga agctcaaagt tggggataga 360 gttgcagtga tgatgagacc tccttcacct cctcaaacta aagctgtagt tgaccctgca 420 atttacgtga aaataagtga ttactacctt gttccgaacg gaaaaggtat gataaaagtt 480 cctaacgatg gtattcctcc agaaaaggcc caatatcttc tttcagtaaa ttcatatcct 540 gtaaaattag tcagagaagt tgatgagaag ttatcctatc tcgctggagt tatactcggt 600 gatgggtata tatcatcgaa tggatactac atctcagcta catttgacga cgaagcttac 660 atggatgcct ttgtctctgt agtctcggac tttatcccta actatgtccc cagtataagg 720 aagaacggag attacacaat tgtaactgtt ggctcgaaga tttttgctga aatgctctca 780 aggatatttg gaataccaag gggcagaaaa tctatgtggg atattccaga cgtagtactt 840 tcaaatgacg atcttatgag atacttcata gctggacttt tcgacgctga tgggtacgta 900 gatgaaaatg ggccctccat agtcctagta acaaagagtg aaaccgtggc aaggaagatt 960 tggtacgttc ttcagaggtt ggggatcata agtacagttt cccgtgtaaa gagcagaggg 1020 tttaaagaag gcgagctgtt cagggtaatt attagtggtg ttgaagatct tgctaaattt 1080 gcaaaattca tacccctacg tcactcaaga aagagggcca aacttatgga gatattaagg 1140 actaagaagc catatcgggg aagaagaact taccgcgtgc cgatatccag tgatatgata 1200 gctcctctcc gtcaaatgtt gggattaact gttgcagagc tgtctaagtt agcgtcttat 1260 tatgcagggg aaaaagtttc tgaaagccta attaggcata tagaaaaggg aagggtcaaa 1320 gagataagac gctctacgct caaggggatt gcccttgctc tccagcagat agctaaagat 1380 gtgggtaacg aagaagcttg ggtgagagcc aagaggcttc aattgatagc tgagggagat 1440 gtttactggg atgaagtcgt aagtgttgag gaagttgatc cgaaggagct tggcattgag 1500 tacgtctatg acctcacggt tgaggacgac cacaattatg tggcaaatgg catactagtc 1560 tcaaac 1566 <210> 39 <211> 522 <212> PRT <213> Pyrococcus furiosus <400> 39 Ala Leu Tyr Asp Phe Ser Val Ile Gln Leu Ser Asn Gly Arg Phe Val 1 5 10 15 Leu Ile Gly Asp Leu Val Glu Glu Leu Phe Lys Lys Tyr Ala Glu Lys             20 25 30 Ile Lys Thr Tyr Lys Asp Leu Glu Tyr Ile Glu Leu Asn Glu Glu Asp         35 40 45 Arg Phe Glu Val Val Ser Val Ser Pro Asp Leu Lys Ala Asn Lys His     50 55 60 Val Val Ser Arg Val Trp Arg Arg Lys Val Arg Glu Gly Glu Lys Leu 65 70 75 80 Ile Arg Ile Lys Thr Arg Thr Gly Asn Glu Ile Ile Leu Thr Arg Asn                 85 90 95 His Pro Leu Phe Ala Phe Ser Asn Gly Asp Val Val Arg Lys Glu Ala             100 105 110 Glu Lys Leu Lys Val Gly Asp Arg Val Ala Val Met Met Arg Pro Pro         115 120 125 Ser Pro Pro Gln Thr Lys Ala Val Val Asp Pro Ala Ile Tyr Val Lys     130 135 140 Ile Ser Asp Tyr Tyr Leu Val Pro Asn Gly Lys Gly Met Ile Lys Val 145 150 155 160 Pro Asn Asp Gly Ile Pro Pro Glu Lys Ala Gln Tyr Leu Leu Ser Val                 165 170 175 Asn Ser Tyr Pro Val Lys Leu Val Arg Glu Val Asp Glu Lys Leu Ser             180 185 190 Tyr Leu Ala Gly Val Ile Leu Gly Asp Gly Tyr Ile Ser Ser Asn Gly         195 200 205 Tyr Tyr Ile Ser Ala Thr Phe Asp Asp Glu Ala Tyr Met Asp Ala Phe     210 215 220 Val Ser Val Val Ser Asp Phe Ile Pro Asn Tyr Val Pro Ser Ile Arg 225 230 235 240 Lys Asn Gly Asp Tyr Thr Ile Val Thr Val Gly Ser Lys Ile Phe Ala                 245 250 255 Glu Met Leu Ser Arg Ile Phe Gly Ile Pro Arg Gly Arg Lys Ser Met             260 265 270 Trp Asp Ile Pro Asp Val Val Leu Ser Asn Asp Asp Leu Met Arg Tyr         275 280 285 Phe Ile Ala Gly Leu Phe Asp Ala Asp Gly Tyr Val Asp Glu Asn Gly     290 295 300 Pro Ser Ile Val Leu Val Thr Lys Ser Glu Thr Val Ala Arg Lys Ile 305 310 315 320 Trp Tyr Val Leu Gln Arg Leu Gly Ile Ile Ser Thr Val Ser Arg Val                 325 330 335 Lys Ser Arg Gly Phe Lys Glu Gly Glu Leu Phe Arg Val Ile Ile Ser             340 345 350 Gly Val Glu Asp Leu Ala Lys Phe Ala Lys Phe Ile Pro Leu Arg His         355 360 365 Ser Arg Lys Arg Ala Lys Leu Met Glu Ile Leu Arg Thr Lys Lys Pro     370 375 380 Tyr Arg Gly Arg Arg Thr Tyr Arg Val Pro Ile Ser Ser Asp Met Ile 385 390 395 400 Ala Pro Leu Arg Gln Met Leu Gly Leu Thr Val Ala Glu Leu Ser Lys                 405 410 415 Leu Ala Ser Tyr Tyr Ala Gly Glu Lys Val Ser Glu Ser Leu Ile Arg             420 425 430 His Ile Glu Lys Gly Arg Val Lys Glu Ile Arg Arg Ser Thr Leu Lys         435 440 445 Gly Ile Ala Leu Ala Leu Gln Gln Ile Ala Lys Asp Val Gly Asn Glu     450 455 460 Glu Ala Trp Val Arg Ala Lys Arg Leu Gln Leu Ile Ala Glu Gly Asp 465 470 475 480 Val Tyr Trp Asp Glu Val Val Ser Val Glu Glu Val Asp Pro Lys Glu                 485 490 495 Leu Gly Ile Glu Tyr Val Tyr Asp Leu Thr Val Glu Asp Asp His Asn             500 505 510 Tyr Val Ala Asn Gly Ile Leu Val Ser Asn         515 520 <210> 40 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 40 Gly His Asp Gly One <210> 41 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 41 Ser Pro Gly Lys One <210> 42 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 42 Ala Leu Tyr Tyr One <210> 43 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 43 Cys Leu Tyr Tyr One <210> 44 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 44 Cys Met Gly Thr One <210> 45 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 45 Met Asp Ile Gln One <210> 46 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 46 Leu Ser Leu Ser Pro Gly Lys 1 5 <210> 47 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 47 Leu Ser Leu Ser Pro Gly 1 5 <210> 48 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 48 Ala Leu Tyr Tyr Phe Ser Glu Ile Gln 1 5 <210> 49 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 49 gcctctccct gtctccgggt gctctgtact acttcagcga gatc 44 <210> 50 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 50 gcctctccct gtctccgggt tgtctgtact acttcagcga gatc 44 <210> 51 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 51 tctccctgtc tccgggtaaa tgtctgtact acttcagcga gatc 44 <210> 52 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 52 cggcgtggag gtgcataatg 20 <210> 53 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 53 acccggagac agggagag 18 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 54 gggtcagcac cagttctttg 20 <210> 55 <211> 476 <212> PRT <213> Pyrococcus horikoshii <400> 55 Gln Cys Phe Ser Gly Glu Glu Val Ile Ile Val Glu Lys Gly Lys Asp 1 5 10 15 Arg Lys Val Val Lys Leu Arg Glu Phe Val Glu Asp Ala Leu Lys Glu             20 25 30 Pro Ser Gly Glu Gly Met Asp Gly Asp Ile Lys Val Thr Tyr Lys Asp         35 40 45 Leu Arg Gly Glu Asp Val Arg Ile Leu Thr Lys Asp Gly Phe Val Lys     50 55 60 Leu Leu Tyr Val Asn Lys Arg Glu Gly Lys Gln Lys Leu Arg Lys Ile 65 70 75 80 Val Asn Leu Asp Lys Asp Tyr Trp Leu Ala Val Thr Pro Asp His Lys                 85 90 95 Val Phe Thr Ser Glu Gly Leu Lys Glu Ala Gly Glu Ile Thr Glu Lys             100 105 110 Asp Glu Ile Ile Arg Val Pro Leu Val Ile Leu Asp Gly Pro Lys Ile         115 120 125 Ala Ser Thr Tyr Gly Glu Asp Gly Lys Phe Asp Asp Tyr Ile Arg Trp     130 135 140 Lys Lys Tyr Tyr Glu Lys Thr Gly Asn Gly Tyr Lys Arg Ala Ala Lys 145 150 155 160 Glu Leu Asn Ile Lys Glu Ser Thr Leu Arg Trp Trp Thr Gln Gly Ala                 165 170 175 Lys Pro Asn Ser Leu Lys Met Ile Glu Glu Leu Glu Lys Leu Asn Leu             180 185 190 Leu Pro Leu Thr Ser Glu Asp Ser Arg Leu Glu Lys Val Ala Ile Ile         195 200 205 Leu Gly Ala Leu Phe Ser Asp Gly Asn Ile Asp Arg Asn Phe Asn Thr     210 215 220 Leu Ser Phe Ile Ser Ser Glu Arg Lys Ala Ile Glu Arg Phe Val Glu 225 230 235 240 Thr Leu Lys Glu Leu Phe Gly Glu Phe Asn Tyr Glu Ile Arg Asp Asn                 245 250 255 His Glu Ser Leu Gly Lys Ser Ile Leu Phe Arg Thr Trp Asp Arg Arg             260 265 270 Ile Ile Arg Phe Phe Val Ala Leu Gly Ala Pro Val Gly Asn Lys Thr         275 280 285 Lys Val Lys Leu Glu Leu Pro Trp Trp Ile Lys Leu Lys Pro Ser Leu     290 295 300 Phe Leu Ala Phe Met Asp Gly Leu Tyr Ser Gly Asp Gly Ser Val Pro 305 310 315 320 Arg Phe Ala Arg Tyr Glu Glu Gly Ile Lys Phe Asn Gly Thr Phe Glu                 325 330 335 Ile Ala Gln Leu Thr Asp Asp Val Glu Lys Lys Leu Pro Phe Phe Glu             340 345 350 Glu Ile Ala Trp Tyr Leu Ser Phe Phe Gly Ile Lys Ala Lys Val Arg         355 360 365 Val Asp Lys Thr Gly Asp Lys Tyr Lys Val Arg Leu Ile Phe Ser Gln     370 375 380 Ser Ile Asp Asn Val Leu Asn Phe Leu Glu Phe Ile Pro Ile Ser Leu 385 390 395 400 Ser Pro Ala Lys Arg Glu Lys Phe Leu Arg Glu Val Glu Ser Tyr Leu                 405 410 415 Ala Ala Val Pro Glu Ser Ser Leu Ala Gly Arg Ile Glu Glu Leu Arg             420 425 430 Glu His Phe Asn Arg Ile Lys Lys Gly Glu Arg Arg Ser Phe Ile Glu         435 440 445 Thr Trp Glu Val Val Asn Val Thr Tyr Asn Val Thr Thr Glu Thr Gly     450 455 460 Asn Leu Leu Ala Asn Gly Leu Phe Val Lys Asn Ser 465 470 475 <210> 56 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 56 His His His His His 1 5 <210> 57 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 57 His His His His His His His His His His 1 5 10 <210> 58 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 58 His Gln His Gln His Gln 1 5 <210> 59 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 59 Met Asp Met Arg Val Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys             20 <210> 60 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 60 Met Asp Met Arg Val Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys             20 <210> 61 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 61 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Gln Leu Trp 1 5 10 15 Leu Ser Gly Ala Arg Cys             20 <210> 62 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 62 Met Asp Met Arg Val Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Ser Gly Ala Arg Cys             20 <210> 63 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 63 Met Asp Met Arg Val Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Asp Thr Arg Cys             20 <210> 64 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 64 Met Asp Met Arg Val Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ala Arg Cys             20 <210> 65 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 65 Met Asp Met Arg Val Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ala Arg Cys             20 <210> 66 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 66 Met Asp Met Arg Val Leu Ala Gln Leu Leu Gly Leu Leu Leu Leu Cys 1 5 10 15 Phe Pro Gly Ala Arg Cys             20 <210> 67 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 67 Met Asp Met Arg Val Leu Ala Gln Leu Leu Gly Leu Leu Leu Leu Cys 1 5 10 15 Phe Pro Gly Ala Arg Cys             20 <210> 68 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 68 Met Asp Met Arg Val Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys             20 <210> 69 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 69 Met Asp Met Arg Val Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys             20 <210> 70 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 70 Met Asp Met Arg Val Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ser Arg Cys             20 <210> 71 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 71 Met Asp Met Arg Val Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ser Arg Cys             20 <210> 72 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 72 Met Asp Met Arg Val Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys             20 <210> 73 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 73 Met Asp Met Arg Val Pro Ala Gln Arg Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ala Arg Cys             20 <210> 74 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 74 Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Gly ala arg cys             20 <210> 75 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 75 Met Asp Met Arg Val Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys             20 <210> 76 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 76 Met Asp Met Arg Val Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys             20 <210> 77 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 77 Met Asp Met Arg Val Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Lys Cys             20 <210> 78 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 78 Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Pro 1 5 10 15 Gly Ser Ser Glu             20 <210> 79 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 79 Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Pro 1 5 10 15 Gly Ser Ser Glu             20 <210> 80 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 80 Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Pro 1 5 10 15 Gly Ser Ser Gly             20 <210> 81 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 81 Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Pro 1 5 10 15 Gly Ser Ser Gly             20 <210> 82 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 82 Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Ile Pro 1 5 10 15 Gly Ser Ser Ala             20 <210> 83 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 83 Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Ile Pro 1 5 10 15 Gly Ser Ser Ala             20 <210> 84 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 84 Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Ser 1 5 10 15 Gly Ser Ser Gly             20 <210> 85 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 85 Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Ser 1 5 10 15 Gly Ser Ser Gly             20 <210> 86 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 86 Met Arg Leu Leu Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Pro 1 5 10 15 Gly Ser Ser Gly             20 <210> 87 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 87 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly             20 <210> 88 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 88 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly             20 <210> 89 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 89 Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly             20 <210> 90 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 90 Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly             20 <210> 91 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 91 Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly             20 <210> 92 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 92 Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Thr 1 5 10 15 Asp Thr Thr Gly             20 <210> 93 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 93 Met Glu Pro Trp Lys Pro Gln His Ser Phe Phe Phe Leu Leu Leu Leu 1 5 10 15 Trp Leu Pro Asp Thr Thr Gly             20 <210> 94 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 94 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly             20 <210> 95 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 95 Met Gly Ser Gln Val His Leu Leu Ser Phe Leu Leu Leu Trp Ile Ser 1 5 10 15 Asp Thr Arg Ala             20 <210> 96 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 96 Met Leu Pro Ser Gln Leu Ile Gly Phe Leu Leu Leu Trp Val Pro Ala 1 5 10 15 Ser Arg Gly              <210> 97 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 97 Met Leu Pro Ser Gln Leu Ile Gly Phe Leu Leu Leu Trp Val Pro Ala 1 5 10 15 Ser Arg Gly              <210> 98 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 98 Met Val Ser Pro Leu Gln Phe Leu Arg Leu Leu Leu Leu Trp Val Pro 1 5 10 15 Ala Ser Arg Gly             20 <210> 99 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 99 Met Asp Met Arg Val Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ser Gly Gly             20 <210> 100 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 100 Met Asp Met Arg Val Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ser Gly Gly Gly             20 <210> 101 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 101 Met Asp Met Arg Val Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ser Gly Gly Gly             20 <210> 102 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 102 Met Asp Met Arg Val Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ser Gly Gly Gly Gly             20 25 <210> 103 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 103 Met Arg Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ser Arg Cys             20 <210> 104 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 104 Met Arg Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ser Gly Gly             20 <210> 105 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 105 Met Arg Arg Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu 1 5 10 15 Trp Phe Pro Gly Ser Arg Cys             20 <210> 106 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 106 Met Arg Arg Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu 1 5 10 15 Trp Phe Pro Gly Ser Gly Gly             20 <210> 107 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 107 Met Arg Arg Arg Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu 1 5 10 15 Leu Trp Phe Pro Gly Ser Gly Gly             20 <210> 108 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 108 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Asp Glu Trp Phe Pro 1 5 10 15 Gly Ser Gly Gly             20

Claims (46)

An isolated or purified expression vector for producing one or more recombinant protein products comprising a single open reading frame insert, wherein the insert is
(a) a signal peptide nucleic acid sequence encoding a signal peptide;
(b) a first nucleic acid sequence encoding a first polypeptide;
(c) a first intervening nucleic acid sequence encoding a first protein cleavage site, wherein the first protein cleavage site is a lon protease gene of Pyrococcus or Pyrococcus or Methanococcus By an intein segment of the klbA gene, or a modified intein segment derived therefrom); And
(d) a second nucleic acid sequence encoding a second polypeptide, wherein said first intervening nucleic acid sequence encoding said first protein cleavage site comprises said first nucleic acid sequence and said second nucleic acid sequence; Operatively located between the nucleic acid sequences; Wherein the signal peptide nucleic acid sequence encoding the signal peptide is operatively located before the first nucleic acid sequence; Wherein said expression vector is capable of expressing a single transcription translation frame polypeptide cleavable at said first protein cleavage site,
An isolated or purified expression vector for producing one or more recombinant protein products comprising a single transcription translation frame insert. The method of claim 1, wherein the first protein cleavage site is Pyrococcus abyssi ), Pyrococcus furiosus ) or Pyrococcus horikoshii ) intein segment of the lon protease gene of OT3; Or intein segments of the klbA gene of Pyrococcus acci, Pyrococcus puriosus or Methanococcus jannaschii ; Or an expression vector provided by a modified intein segment, each derived from them. The expression vector of claim 1, wherein the intein segment or modified intein segment encodes a penultimate residue at the end of lysine, serine but not histidine. The expression vector of claim 1, wherein said intein segment or modified intein segment is capable of cleaving said first polypeptide but not fully connecting it to said second polypeptide. The intetan segment of claim 1, wherein the first protein cleavage site comprises a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 4, 6, 7, 55, 35, 37, and 39 An expression vector provided by one modified intein segment. The expression vector according to any one of claims 1 to 5, wherein the first polypeptide and the second polypeptide can multimericly assemble. The expression vector of claim 1, wherein at least one of the first polypeptide and the second polypeptide is capable of extracellular secretion. 8. The expression vector of claim 1, wherein at least one of the first polypeptide and the second polypeptide is of mammalian origin. 9. The method of claim 1, wherein the first polypeptide comprises an immunoglobulin heavy chain or functional fragment thereof, and the second polypeptide comprises an immunoglobulin light chain or functional fragment thereof, Wherein said first polypeptide is on top of said second polypeptide. The expression vector of claim 1, which comprises only one of the signal peptide nucleic acid sequences. The method of claim 1, further comprising: a third nucleic acid sequence encoding a third polypeptide, and a second intervening nucleic acid sequence encoding a second protein cleavage site; Wherein the second intervening nucleic acid sequence and the third nucleic acid sequence are operatively located after the second nucleic acid sequence in this order. The method of claim 1, wherein the first and second polypeptides comprise TNF-α (tumor necrosis factor-alpha), erythropoietin receptor, RSV, EL / selectin, interleukin-1, Antigen selected from the group consisting of interleukin-12, interleukin-13, interleukin-18, interleukin-23, interleukin-33, CD81, CD19, IGF1, IGF2, EGFR, CXCL-13, GLP-1R, prostaglandin E2 and amyloid beta An expression vector comprising a functional antibody or other antigen recognition molecule with antigen specificity directed against binding. The method of claim 1, wherein the first and second polypeptides comprise a pair of immunoglobulin chains from an antibody of D2E7, EL246, ABT-007, ABT-325, or ABT-874. Expression vector. The immunoglobulin heavy chain according to any one of claims 1 to 13, wherein the first and second polypeptides are from analogous segments of D2E7, EL246, ABT-007, ABT-325, ABT-874, or other antibodies. Or an expression globulin light chain segment, each independently selected. The expression vector of claim 1, wherein the vector further comprises a promoter regulatory component for the insert. The expression vector of claim 15, wherein the promoter regulatory component is inducible or constitutive. The expression vector of claim 15, wherein the promoter regulatory component is tissue specific. The expression vector of claim 15, wherein the promoter comprises an adenovirus major late promoter. A host cell comprising the vector according to any one of claims 1 to 18. The host cell of claim 19, wherein the host cell is a prokaryotic cell. The host cell of claim 20, wherein the host cell is Escherichia coli . The host cell of claim 19, wherein the host cell is a eukaryotic cell. The host cell of claim 22, wherein the eukaryotic cell is selected from the group consisting of protozoal cells, animal cells, plant cells and fungal cells. The host cell of claim 23, wherein the eukaryotic cell is an animal cell selected from the group consisting of mammalian cells, avian cells, and insect cells. The host cell of claim 24, wherein the host cell is a mammalian cell line. The host cell of claim 24, wherein the host cell is a CHO cell or a dihydrofolate reductase-deficient CHO cell. The host cell of claim 24, wherein the host cell is a COS cell or HEK cell. The host cell of claim 23, wherein the host cell is a yeast cell. The host cell of claim 28, wherein the yeast cell is Saccharomyces cerevisiae . The host cell of claim 24, wherein the host cell is Spodoptera frugiperda Sf9 insect cell. 20. A method for producing recombinant polyprotein or a plurality of proteins, comprising culturing the host cell according to claim 19 in a culture medium under conditions sufficient to permit expression of the vector protein. 32. The method of claim 31, further comprising recovering and / or purifying the vector protein. 33. The method of any one of claims 31-32, wherein the plurality of proteins can multimeric assemble. 34. The method of any one of claims 31-33, wherein the recombinant polyprotein or a plurality of proteins is biologically functional and / or therapeutic. A recombinant product, which is an immunoglobulin protein or functional fragment thereof, assembled antibody, or other antigen recognition molecule, comprising culturing a host cell according to claim 19 under conditions sufficient to produce a recombinant product in a culture medium. How to produce. A protein produced according to the method according to any one of claims 31 to 35. 37. Polyprotein produced according to the method according to any one of claims 31 to 36. 38. An assembled immunoglobulin produced according to the method of any one of claims 31-37; Assembled other antigen recognition molecule; Or individual immunoglobulin chains or functional fragments thereof. The method of claim 38, wherein the tumor necrosis factor-α, erythropoietin receptor, RSV, EL / selectin, interleukin-1, interleukin-12, interleukin-13, interleukin-18, interleukin-23, interleukin-33, CD81, CD19 Immunoglobulins, which have the ability to affect or contribute to specific antigen binding to IGF1, IGF2, EGFR, prostaglandin E2, CXCL-13, GLP-1R, or amyloid beta; Other antigen recognition molecules; Or individual immunoglobulin chains or functional fragments thereof. The immunoglobulin or functional fragment thereof of claim 39, wherein the immunoglobulin is D2E7 or ABT-874 or the functional fragment is a fragment thereof, respectively. 41. A pharmaceutical composition comprising a therapeutically effective amount of a protein according to any one of claims 36-40 and a pharmaceutically acceptable carrier. The expression vector of claim 1, further comprising a nucleic acid sequence encoding a tag. The expression vector of claim 1, wherein the intervening nucleic acid sequence further encodes a tag. An expression vector, a host cell having the vector, a vector expression product, a pharmaceutical composition, and / or a method of making or using any of the above, wherein the vector is a compound according to any one of claims 1 to 18. And a method according to any one of the foregoing, an expression vector, a host cell having the vector, a vector expression product, a pharmaceutical composition and / or any one of the foregoing. . 45. The vector, host cell, vector expression product, pharmaceutical composition and / or method of manufacture of claim 44, wherein the encoded light chain signal peptide is a kappa light chain signal peptide selected from the group consisting of A17, A18, A19, A26 and H2G. Or how to use. 45. The vector, host cell, vector expression product, pharmaceutical composition and / or method of manufacture or use of claim 44, wherein the encoded light chain signal peptide is VKII kappa light chain signal peptide A18, SEQ ID NO: 82 (amino acid sequence MRLPAQLLGLLMLWIPGSSA). .

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